Regulation of the Expression of Aminoacyl-tRNA Synthetases and Translation Factors
Chapter
91
MARIANNE GRUNBERG-MANAGO
The translational apparatus is the major machinery of the cell. In addition to ribosomes, tRNA, and mRNA, it includes the following numerous components: monovalent and divalent ions, nucleotides and proteins such as tRNA-modifying enzymes, aminoacyl-tRNA synthetases, and proteins transiently associated with the ribosomes (initiation, elongation, and release factors [IF, EF, and RF, respectively]). The components of the machinery are coded for by more than 200 genes, and a large part of cellular energy is committed to the synthesis of the translational apparatus. In this chapter, I consider our current knowledge of the gene organization and the expression of aminoacyl-tRNA synthetases and proteins associated with ribosomes during the cycle of polypeptide synthesis. The genes for ribosomal components and tRNAs are the subject of chapter 90. The synthesis of aminoacyl-tRNA synthetases and the protein factors associated with ribosomes are in most cases coordinated with the synthesis of ribosomal components. The mechanism of this coordination remains unknown and is not discussed in detail in this review. Most of the work reported here deals with specific controls of the expression of the various genes considered.
The aminoacyl-tRNA synthetases play a central role in protein biosynthesis by catalyzing the attachment of amino acids to the 3' ends of their cognate tRNAs. The precision of aminoacylation contributes to the accuracy of protein biosynthesis. In addition to their crucial role in protein synthesis, the aminoacyl-tRNA synthetases are engaged in a number of regulatory processes via their product, the charged tRNA. The concentration of amino acid-charged tRNA might regulate the elongation rate of protein synthesis (137). The control of the expression of amino acid biosynthetic operons by the degree of in vivo tRNA aminoacylation is well documented. Another phenomenon, the pleiotropic stringent response, operates when tRNA aminoacylation is diminished in wild-type (relA +) strains of Escherichia coli and inhibits rRNA and tRNA synthesis as well as the synthesis of some other macromolecules involved in translation (see below).
In addition, aminoacyl-tRNA synthetases participate in a number of other reactions, such as posttranslational N-terminal aminoacylation (52) and amino acid transport (176, 198, 222). Diadenosine 5'-5'''-P1,P4-tetraphosphate (AppppA) is formed as a by-product of the amino acid activation step and may be an important pleiotropic activator (14, 148, 274, 287).
It is obviously important to our knowledge of cell physiology to understand the mechanism(s) which controls the cellular levels of aminoacyl-tRNA synthetases.
A single aminoacyl-tRNA synthetase for each amino acid has been found in E. coli (136, 166, 253) except that two species of lysyl-tRNA synthetase have been characterized (107). The cellular abundance of the different aminoacyl-tRNA synthetases is quite similar; at 37°C, around 500 molecules of enzyme per genome appear in cells grown on minimal medium with glucose as the sole carbon source, and 800 molecules per genome appear in cells grown in rich medium. These amounts are about 20 times less than the number of ribosomes (19, 187). Under normal conditions, the different tRNAs are 80 to 85% aminoacylated. The activities of the different synthetases in vivo (moles of tRNA charged per second per mole of synthetase) correspond to the rate of incorporation of the cognate amino acids into protein (126). Thus, it is thought that the aminoacyl-tRNA synthetases are not limiting for protein synthesis.
Aminoacyl-tRNA synthetase levels are regulated in a variety of ways. The different molecular mechanisms discovered thus far are described below.
Metabolic Regulation.
Metabolic regulation affects all of the aminoacyl-tRNA synthetases studied to date (specifically, arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, valine, methionine, and serine) (187, 226). This type of regulation results in a two- to threefold increase in the level of enzyme for each fivefold increase in the growth rate. The increase is less than that observed with ribosomes but corresponds to that of elongation factor EF-Tu and the initiation factors. Thus, in general, the aminoacyl-tRNA synthetase/ribosome ratio decreases progressively at high growth rates, whereas the synthetase/EF-Tu ratio is virtually independent of growth rate.
Regulation as a function of growth rate occurs at the transcriptional step for valyl (226), tryptophanyl (93, 94, 95), and glutaminyl-tRNA synthetases (38). The indication that growth rate control occurs directly at the transcriptional level comes from experiments in which steady-state mRNA measurements were made under different growth rate conditions and showed an increase of aminoacyl-tRNA synthetase mRNA with increasing growth rate. However, as was pointed out elsewhere (220), the increase in mRNA levels could be the consequence of an increase in translation enhancing mRNA stability and thus the steady-state amount of mRNA. Changes in growth rate can differently affect the initiation of translation from individual E. coli mRNAs (125). No molecular mechanism for metabolic regulation has been proposed. In the few cases in which a specific control mechanism has been shown to operate on synthetase expression, the relationship between the specific control and the more general growth control was investigated (220; see below). In the case of thrS, growth rate regulation may be caused by changes in the cellular tRNAThr concentration in a way quite similar to that with which rRNA synthesis rates modulate the expression of the ribosomal protein operons (M. M. Comer et al., unpublished data).
Derepression upon Amino Acid Starvation.
Superimposed on metabolic regulation is a regulatory response of individual synthetase genes to limiting amounts of the cognate amino acid. At least half of the aminoacyl-tRNA synthetases are synthesized more rapidly in cells starved for their respective amino acids (186; for reviews see references 179 and 188). The derepression can be observed only in appropriate E. coli bradytrophs (leaky auxotrophs) or by use of amino acid analogs (156, 162, 186). The response is variable; seven aminoacyl-tRNA synthetase genes (thrS, hisS, leuS, metG, proS, serS, and valS) are transiently derepressed, and three (pheST, argS, and ileS) are permanently derepressed. In all cases in which derepression is found, addition of the amino acid after starvation represses the expression. In the case of two synthetase genes, hisS of Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) and metG of E. coli, the depression is due to deacylation of the specific tRNA. The role of histidyl-tRNA in the synthesis of histidyl-tRNA synthetase was demonstrated in two mutant strains of S. typhimurium (163, 164). The hisR mutant (mutation in the gene for tRNAHis) has only 52% of the histidyl-tRNA synthetase of the wild-type strain, whereas the hisS mutant has a histidyl-tRNA synthetase with an increased Km for histidine. Histidine-restricted growth results in an increase in the rate of formation of histidyl-tRNA synthetase in the wild type and in both mutant strains. However, addition of histidine to the growth medium when the synthetase is derepressed causes a repression in the wild-type strain but not in the hisR mutant. The repression is also seen in the hisS mutant when histidine is added to yield a concentration higher than the Km of the mutant enzyme. These data indicate that histidyl-tRNA, not the free amino acid, is involved in regulation of the expression of the synthetase. The same conclusion was reached for methionyl-tRNA synthetase; a decrease in the in vivo acylation level of tRNAMet induced by the addition of methionyl-adenylate led to a specific derepression of metG. The level of derepression was proportional to the amount of deacylated tRNAMet (34).
Stringent Control.
When enteric bacteria are subjected to amino acid deprivation, they adjust their metabolism by ceasing to make rRNA and tRNAs. The stringent response is governed by the gene relA. A mutation in relA causes relaxed control, allowing the continued production of stable RNAs under conditions of amino acid deprivation. The extent of aminoacylation of tRNAs plays a central role in this response and may function by controlling the level of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). The effect of amino acid starvation on the synthesis of aminoacyl-tRNA synthetases was studied by using a temperature-sensitive E. coli valyl-tRNA synthetase strain to induce the stringent response (15, 194). After temperature shiftup, the rates of synthesis of 10 aminoacyl-tRNA synthetases (arginyl-, glutamyl-, glutaminyl-, glycyl-, isoleucyl-, leucyl-, lysyl-, phenylalanyl-, valyl-, and threonyl-tRNA synthetases were examined. Except for arginyl- and maybe glutaminyl-tRNA synthetases, the synthesis of which was moderately repressed, all were unaffected. It appears that the expression of aminoacyl-tRNA synthetase genes is not clearly stringently regulated.
Temperature Response.
Temperature shifts affect the rates of synthesis of at least 10 aminoacyl-tRNA synthetases. When a culture is shifted from 28 to 42°C, a transient, approximately twofold decrease results. A similar response is observed for the elongation factors EF-G and EF-Ts but not for EF-Tu, whose expression does not vary much with temperature (149). An exception to the general response of aminoacyl-tRNA synthetases to temperature occurs with lysyl-tRNA synthetase (see below).
Subunit structures and molecular weights are surprisingly variable among the different synthetases (244). The different subunit structures are of the α, α 2, α 4, and α 2β2 types. The genes for the synthetase subunits are always unique except that lysyl-tRNA synthetase is coded for by two genes, lysS and lysU (61, 269).
The genes for all aminoacyl-tRNA synthetases have been cloned, sequenced, and mapped on the chromosome (Fig. 1). Except for the threonyl-tRNA synthetase gene thrS and the phenylalanyl-tRNA synthetase gene pheST, which are grouped at 37.4 min (65, 206), and the aspartyl-tRNA synthetase gene aspS and the arginyl-tRNA synthetase gene argS, which are located at 41 min, the genes are scattered throughout the whole chromosome. There is no correlation between the orientation of transcription of different synthetase genes and the direction of replication. There is also no correlation between the positions of the synthetase genes and tRNA genes except for the gene encoding glutamyl-tRNA synthetase (gltX).
The nucleotide sequences of the regulatory regions of all the synthetase genes have been determined, and in most cases, the transcription initiation sites are known (220).
Phenylalanyl- and glycyl-tRNA synthetases are the only synthetases composed of two different subunits (278). Both are tetramers of the α 2 β 2 structural type (278). The genes for the two different subunits are grouped on the chromosome (134) and are expressed as an operon in which the promoter-proximal gene codes for the small subunit.
The synthesis of aminoacyl-tRNA synthetases is subject to control by a variety of genetic mechanisms. In attempts to find regulatory genes or elements, efforts have been directed at isolating and characterizing mutations affecting these control mechanisms. Control mutants can be found by selecting thermoresistant pseudorevertants of a thermosensitive mutant in the structural gene of a given synthetase. The mutants isolated in this way can be classified in the following four categories.
Pseudoreversion due to tRNA Genes.
Thermoresistant pseudorevertants of a thermosensitive glnS mutant increase 
levels. glnT increases the level of (178). trnA (glnU) increases the levels of and 
and of several other tRNA species (37). This reversion phenotype could be explained by protection of the mutated enzyme by an excess of its substrate. The same type of phenotypic reversion is seen in the case of phenylalanyl-tRNA synthetase (29, 30, 246). A multicopy plasmid carrying either of the structural genes for tRNAPhe (pheU or pheV) causes some strains carrying thermosensitive alleles of pheS or pheT to grow at high temperature.
Pseudoreversion due to Ribosomal Protein Mutants.
Thermoresistant pseudorevertants of thermosensitive alaS (24) and valS (284) strains are located in the gene for ribosomal protein S5 or S8 (alaS) and in the gene for S20 (valS). A possible explanation for this surprising phenomenon is the fact that the creation of amino acid-charged tRNA pools must be balanced with translation rates. Mutant aminoacyl-tRNA synthetases are unable to charge their cognate tRNAs at the rate at which they are utilized, thereby reducing the level of aminoacylation and slowing the rate of protein synthesis. When another mutation which causes a reduction in the overall rate of protein synthesis is introduced into the translational machinery, the requirement for aminoacyl-tRNA is reduced and the balance between aminoacylation and the translation rate is restored.
Pseudoreversion due to Elevated Levels of Aminoacyl-tRNA Synthetase Caused by Mutations of the Operator or Promoter Type.
Mutations of the operator or promoter type map closely to the enzyme structural gene and are cis dominant. For example, the leuX mutation results in elevated levels of leucyl-tRNA synthetase (143), and the serO mutation results in high levels of seryl-tRNA synthetase (41). In the serO strain, the normal dependence of seryl-tRNA synthetase expression on changes in exogenous serine concentration was not observed. The strain is permanently derepressed for seryl-tRNA synthetase formation, which suggests that the mutation is not in the promoter region but in the region analogous to an operator. The valX mutation also results in an increased level of valyl-tRNA synthetase (9). The increased levels are due to new enzyme formation rather than to a decreased rate of protein degradation, but the molecular mechanism has not yet been elucidated.
Pseudoreversion due to Elevated Levels of Aminoacyl-tRNA Synthetase Caused by Unlinked trans-Acting Mutations.
Four mutations which are responsible for an elevated level of the corresponding thermolabile synthetases have been identified: glnR (37), leuY (142, 143), and leuR and serR (262). In addition, a gene shown to affect the expression of isoleucyl-tRNA synthetase, ilvU, was found in the vicinity of leuY (20), but the relationship between this gene and leuY is unknown. The product of leuY appears to be a protein, because the mutation has been identified as a nonsense mutation: introduction of a specific suppressor tRNA into leuY strains prevents expression of the temperature-resistant phenotype characteristic of leuY. However, the roles of these gene products are not clear, because these mutations are incapable of increasing the expression of wild-type synthetase. A major question still to be resolved is whether the products of these regulatory genes increase the rate of synthesis of the aminoacyl-tRNA synthetases or whether they slow down the rate of their degradation in the cell.
Conclusions.
Although molecular mechanisms for the action of the four regulatory genes described above are not yet apparent, a few conclusions can be drawn.
In all of the known regulatory mutants, a particular mutation exerts an effect on only one particular aminoacyl-tRNA synthetase, and to date, no mutations affecting the control of all or a few aminoacyl-tRNA synthetase genes have been found. Besides the metabolic control, no data demonstrate the existence of coordinate control for any of the synthetases except that a common regulation of the levels of the aminoacyl-tRNA synthetases for the three branched-chain amino acids has been reported (124).
The genes for the aminoacyl-tRNA synthetases which have been studied are not coregulated with those of their cognate tRNAs (263). This lack has been shown for glutaminyl-tRNA synthetase (37), leucyl-tRNA synthetase (142), and phenylalanyl-tRNA synthetase (64, 263). However, under threonine and isoleucine deficiency, which leads to a derepression of the respective synthetase, the concentrations of isoaccepting fractions of the cognate tRNA species were changed (263).
It is also known that the levels of aminoacyl-tRNA synthetases (those for leucine, valine, isoleucine, arginine, and threonine) are not correlated with the levels of three tRNA- modifying enzymes (the tRNA methyl-5-uridine, methyl-1-guanosine, and methylaminomethyl-2-thiouridine methyltransferases) during different steady-state growth conditions (194).
Physiological and genetic studies have not yet elucidated the mechanisms involved in the control of expression of the aminoacyl-tRNA synthetase genes. Instead, studies at the molecular level, made easier through recombinant DNA techniques, have shed considerable light on the problem and are reviewed here.
Autoregulation by Transcriptional Repression (Alanyl-tRNA Synthetase).
Alanyl-tRNA synthetase is a tetramer composed of one type of 95-kDa subunit (215, 216). In vitro transcription studies which used a DNA restriction fragment containing the promoter and part of the alaS structural gene showed that alanyl-tRNA synthetase (in micromolar concentration) specifically represses transcription of its own gene. The histidine operon DNA was used as a control, and transcription of the histidine leader transcript was not affected by alanyl-tRNA synthetase. Transcriptional repression by alanyl-tRNA synthetase is greatly enhanced by elevated (millimolar) concentrations of alanine; alanine in the absence of alanyl-tRNA synthetase has no effect. Because alanyl-tRNA synthetase specifically affects transcription, it may directly interact with the DNA template. Experiments in protection against DNase digestion were carried out with restriction fragments containing the transcription initiation region. Alanyl-tRNA synthetase protects two specific nucleotide sequences against digestion by DNase I. A palindromic sequence is found within each of the protected sequences flanking the transcription initiation site (216).
The intracellular concentration of alanyl-tRNA synthetase in cells growing in rich medium is about 0.4 μM (214), and at this concentration there is little, if any, autogenous repression in vitro in the absence of alanine. The intracellular concentration of alanine in exponentially growing cells is between 1 and 5 mM (198). This high concentration correlates with the high Km for alanine (1 mM), which is substantially higher than the Kms for other synthetases. An in vitro concentration of 1.5 mM alanine causes a 20-fold repression in the presence of 1 μM alanyl-tRNA synthetase. These results indicate that it is reasonable to expect that alanyl-tRNA synthetase autogenous regulation might be influenced or controlled by the intracellular alanine concentration. Because transcription experiments were carried out in the presence of ATP, it could not be determined whether the effector of the control is a complex of the synthetase with alanine, alanine plus ATP, or alanyl-adenylate. Unfortunately, the expected derepression of alanyl-tRNA synthetase synthesis upon alanine starvation was never shown to occur, and no in vivo data have been presented to support the model, which is well documented in vitro.
Regulation by Attenuation (Phenylalanyl-tRNA Synthetase).
Phenylalanyl-tRNA synthetase is tetramer (α 2 β 2) composed of two types of subunit (α = 37 kDa; β = 87 kDa) (63). The genes pheS and pheT, encoding the small and large subunits, respectively, are adjacent at 37.4 min on the E. coli chromosome and are cotranscribed (43, 63, 202). They are situated in a cluster of genes containing thrS, coding for threonyl-tRNA synthetase; infC, coding for initiation factor IF3; rpmL and rplT, coding for ribosomal proteins L35 and L20, respectively; and himA, coding for the large subunit of the host integration factor required for the site-specific integration recombination of λ (for a review, see reference 87) (Fig. 2). The order of the genes is thrS infC rpmL rplT pheS pheT himA, and all are transcribed in the same direction from thrS to himA. The DNA sequence of this entire cluster has been determined. Sequence determination and in vitro transcription indicate a promoter situated 368 bp in front of pheS. Downstream from this promoter and in front of pheS, a putative open reading frame is found to code for a 14-residue peptide containing 5 phenylalanine residues, of which 3 are consecutive (65). The corresponding leader mRNA can fold into several alternative structures, one of which is similar to a Rho-independent terminator (Fig. 2, stem 3/4). However, an alternative RNA conformation (Fig. 2, stem 2/3) could also be formed, precluding the formation of stem 3/4. These features are characteristic of the attenuation mechanism which regulates the expression of many amino acid biosynthetic operons (see chapter 81 in this volume).
The regulation of the pheST operon has been studied in great detail in vitro and in vivo. As mentioned above, physiological studies have shown that the expression of phenylalanyl-tRNA synthetase is derepressed around 2.5-fold in a phenylalanine bradytroph upon phenylalanine starvation (186). This derepression involves de novo synthesis of the enzyme.
The regulation of the pheST operon has been studied in vivo by using gene fusions between pheST and lacZ (261). In most cases, the operon fusions had a regulatory region of the pheST operon fused to a DNA fragment carrying the lacZ gene with its own translational initiation signals. The fusions were cloned in a λ phage, and single copies of λ were integrated into the E. coli chromosome by lysogenization. With such fusions, β-galactosidase activity measures transcription from the pheST promoter into lacZ. The pheST operon is derepressed under conditions in which the cellular concentration of phenylalanyl-charged tRNA is decreased. The concentration of tRNAPhe was varied by using either a host strain carrying a mutated phenylalanine-tRNA synthetase with a high Km for phenylalanine (this strain is bradytrophic for phenylalanine) or a host strain carrying a thermosensitive phenylalanyl-tRNA synthetase. In the case of the phenylalanine bradytroph, the level of β-galactosidase activity increases when the growth medium is limiting for phenylalanine. In the case of the thermosensitive mutation, β-galactosidase activity increases upon growth at elevated temperatures, whereas in the control wild-type strain, no increase is observed. These results indicate that transcription into the pheST structural genes is dependent on the level of aminoacylation of tRNAPhe.
The pheST operon is also derepressed in strains carrying a mutant allele of the miaA (or trpX) gene, the product of which modifies tRNAPhe and tRNATrp, among other tRNAs. The miaA mutation results in the absence of the isopentenyl modification of the adenine adjacent to the anticodon of some tRNAs. The unmodified base alters the translational properties of the tRNA. A mutation in miaA causes derepression of the trp and pheA operons (57, 78, 285), which are known to be controlled by an attenuation mechanism.
The attenuation control of the pheST operon was further documented by in vitro transcription experiments; 90% of the transcription products initiated at the pheST promoter terminate at the Rho-independent terminator situated in front of pheS. However, long runoff transcripts proceeding through the terminator and covering the pheS structural gene were also observed. No other transcription initiation site could be detected between the terminator and the pheS structural gene.
On the basis of in vivo results, sequence determination, and in vitro transcription, the attenuation model indicated in Fig. 2 was proposed for the expression of the pheST operon (65). When the leader mRNA is either inefficiently translated or not translated at all, the terminator structure (stem 3/4) is favored. A lack of phenylalanine-charged tRNA results in stalling of the ribosome at phenylalanine codons of the leader peptide. This allows formation of the antiterminator structure (stem 2/3) and transcription of the pheST structural genes by the RNA polymerase. For details, see the legend to Fig. 2.
To investigate the functions of different segments of the pheST attenuator, a series of insertions, deletions, or point mutations in the pheST leader region was constructed in vitro and introduced into the pheST-lacZ operon fusion carried by a λ phage. The effect of the deletions and mutations on the expression of β-galactosidase was compared in wild-type and miaA mutant backgrounds (161, 258). The results obtained led to the following conclusions. First, the localization of a strong promoter following an efficient terminator in the pheS leader region agrees with in vitro results. In addition to transcription from the pheST promoter, 30% of the transcripts covering the pheST operon come from the upstream gene rplT, the terminator of which is very inefficient. Second, the presence of the attenuator as a whole is essential to the regulation of the operon; deletions of the entire attenuator or mutations in segment 3 cause derepression of pheST expression. Third, the role of alternative structure stem 2/3 as an antiterminator was demonstrated by mutations in segment 2 that resulted in uncontrolled termination, i.e., superattenuation. Fourth, the necessity for the tight coupling between transcription and leader mRNA translation was also demonstrated by reducing the efficiency of translation of the leader peptide or by altering the distance between segment 2 and the stop codon of the leader peptide by insertion of a series of nucleotides. In both cases, derepression occurred. A maximum derepression occurred when the distance between the leader peptide stop codon and segment 2 was increased by 11 nucleotides. A likely explanation for this derepression is that a ribosome located on the last codon of the leader peptide is unable to prevent the formation of the antiterminator structure by steric hindrance when the distance between the leader peptide stop codon and segment 2 is too great. The insertions uncouple the formation of the stem 2/3 RNA structure from the translation of the leader peptide.
In conclusion, studies with fusions carrying the modified leader mRNA give a better insight into the pheST regulatory process and definitively establish that the control of expression of the pheST operon is mediated by an attenuation mechanism. However, the pheST operon is different from the amino acid biosynthetic operons controlled by attenuation, since it is composed of genes essential for bacterial growth (the absence of the operon cannot be compensated for by supplementary nutrients in the growth medium). Even in the repressed state, phenylalanyl-tRNA synthetase must be present at a relatively high cellular concentration (about 1 μM). Thus, the level of transcription traversing the terminator of the attenuator must be appreciable. One can estimate that under normal growth conditions at 37°C in glucose, about 15% of the RNA polymerases read through the terminator into the structural genes of the operon. It appears that the high level of termination at the attenuator is compensated for by the strength of the pheST promoter and by the presence of additional transcripts originating from upstream genes.
Autoregulation at the Translational Step (Threonyl-tRNA Synthetase).
Threonyl-tRNA synthetase is a dimer composed of two identical subunits of 73.9 kDa each, the structural gene of which, thrS, is located at 37.4 min on the E. coli chromosome in the vicinity of the pheST operon (103) (see above). The thrS gene is expressed from a transcription initiation site located 162 bp upstream of its structural gene (151) and is separated from the downstream gene, infC, by only three nucleotides (160). mRNA species covering both genes have been observed (27).
Earlier physiological studies showed that thrS expression is transiently derepressed about two- or threefold during starvation for threonine (6, 263). A multicopy plasmid carrying thrS as well as the pheST operon overproduces threonyl-tRNA synthetase about 10-fold and overproduces phenylalanyl-tRNA synthetase about 100-fold (64, 203). Although the overproduction of each of the two synthetases differs by an order of magnitude, their mRNA levels are similar (204). This suggests that thrS is posttranscriptionally regulated.
Threonyl-tRNA synthetase gene expression has been studied in a cell-free protein-synthesizing system programmed with a plasmid DNA carrying the genes thrS, infC, rpmL, rplT, pheS, and pheT. The initial rate of synthesis of threonyl-tRNA synthetase was markedly reduced by the addition of threonyl-tRNA synthetase to the assay but not by the addition of phenylalanyl- or tyrosyl-tRNA synthetase. The inhibition reached 90% with 2 μM physiological concentrations of the enzyme. However, the synthesis of thrS mRNA in the cell-free protein-synthesizing system, as measured by hybridization with specific DNA probes, was not affected by the addition of 2 μM threonyl-tRNA synthetase. This again suggested posttranscriptional control of thrS gene expression (153).
The regulation of thrS expression in vivo was studied by using several thrS-lacZ fusions cloned in λ and integrated as single copies in the E. coli chromosome (27, 259). A protein fusion was constructed such that the N-terminal 335 amino acids coded for by thrS were fused in phase to amino acid 7 of β-galactosidase. The amount of β-galactosidase activity synthesized (under the control of both the thrS promoter and its translational signals) from such a thrS-lacZ protein fusion decreased if threonyl-tRNA synthetase was overproduced in trans from a plasmid carrying thrS.
By using thrS-lacZ translational fusions, mutations that increase the expression of thrS and abolish the negative control were isolated (254). These mutations were all found by DNA sequencing to lie 5' of the structural gene downstream of the transcription initiation site near the Shine-Dalgarno sequence between nucleotides –10 and –50 (A of the initiation triplet AUG is called +1). By analogy with transcriptionally regulated systems, the site of these mutations was called the operator of thrS. The majority of these mutations occurred at G (–32) and U (–31). Detailed probing experiments of the thrS in vitro transcripts with chemical and enzymatic probes showed that thrS leader mRNA folds into four well-defined domains (175) (Fig. 3).
Domain 1 contains the Shine-Dalgarno sequence and the translational initiation codon. Domain 2 is a stem-loop structure containing a sequence corresponding to the threonine-tRNA anticodon CGU in a 7-base loop and sequence similarities with the anticodon stem of several threonine-tRNA isoacceptors (Fig. 3). Domain 3 is not structured, and domain 4 is folded in a very stable structure which has similarities to the acceptor arm of tRNAThr. It contains an ACCA sequence with A-107 homologous to the 3'-terminal A of tRNAThr and located at an equivalent distance from the G-112–C-89 and U-115–A-76 base pairs conserved in the four E. coli tRNAThr isoacceptors (Fig. 3).
A thorough study of the thrS operator, based on multiple selections and mutant constructions (point mutations or deletions), showed that the four domains are structurally independent and correspond to different functional domains (23). Domain 1, which contains the Shine-Dalgarno sequence, is mainly involved in binding the ribosome. Domain 2 and 4, which display analogies with specific domains of tRNAThr, are protected by ThrRS (175, 235). Domain 2, which contains the anticodon-like arm, is the major recognition site of the synthetase (see below). Domain 4 is also directly involved in control, although the sequence analogy between the acceptor arm of tRNAThr and domain 4 of mRNA appears to be fortuitous and has no functional significance.
The point mutations which affect regulation are located in the hairpin loop of domain 4. This placement is consistent with the fact that ThrRS strongly shields the hairpin loop of domain 4. However, in contrast to the mutations in domain 2, none of the mutations in domain 4 completely eliminate control. Domain 4 is therefore not essential but contributes to stabilization of the operator-ThrRS complex.
The third upstream domain, although essential in thrS expression and control, shows no particular nucleotide specificity. It can be point mutated or even inverted without affecting control. However, deletions of more than about 10 of the 23 nucleotides of the whole domain affect expression and regulation of thrS. The domain may thus serve as a communicator between domains 2 and 4.
Proof that the resemblance between domain 2 and the anticodon arm of tRNAThr reflects an authentic functional similarity comes from a number of different experiments. The best evidence is that derived from tRNA identity rules(193). In the case of both E. coli tRNAThr and E. coli tRNAMet, the anticodon is the major determinant of aminoacylation specificity (245). The replacement of CGU of the anticodon-like sequence of the thrS operator by CAU (a single nucleotide change, G to A) is sufficient to abolish control by threonyl-tRNA synthetase and establish control by methionyl-tRNA synthetase in vivo (79).
Further evidence took advantage of the fact that the repressor is an enzyme. An RNA transcript of the leader region of thrS competed with tRNAThr for aminoacylation with ThrRS. The dissociation constant of the wild-type mRNA-ThrRS complex is close to that of the tRNAThr-ThrRS complex (234).
The in vivo identity switch between ThrRS and MetRS control of thrS expression was also shown in vitro. The mutated CAU operator acts as a competitive inhibitor of tRNAMet for methionine aminoacylation catalyzed by E. coli MetRS and no longer for threonine aminoacylation by ThrRS of tRNAThr. This demonstrates that the anticodon-like sequence is one major determinant for the identity of the operator and the specificity of the regulation. The possibility of switching control from ThrRS to MetRS and the fact that the thrS leader mRNA is a competitive inhibitor of tRNA aminoacylation are direct evidence for the existence of molecular mimicry between the translational operator and tRNA. They strongly suggest that the operator and the tRNA bind to the same or overlapping sites in the enzyme and that all the recognition elements for ThrRS binding are present in the thrS operator region. This possibility is also supported by the isolation of ThrRS mutants that have parallel effects on the autoregulation and aminoacylation of tRNAThr (255). Therefore, the mRNA should be able to adopt a tRNA-like structure. The graphic modeling data (to be published) indicate that the loop of domain 3 connecting anticodon-like domain 2 to domain 4 helps place these two domains in the correct orientation relative to each other. This orientation confers a more relaxed conformation on the mRNA relative to the tRNA, which facilitates mRNA adaptation to its cognate aminoacyl-tRNA synthetase as well as its interaction with the ribosome.
The effect of synthetase binding to its mRNA target site on the recognition by the ribosome of its initiation site was studied with the reverse transcriptase primer extension inhibition technique called toeprinting (98). Although ribosomes bind extremely well in vitro to a wild-type thrS leader transcript, this binding is completely inhibited by threonyl-tRNA synthetase in physiological concentrations. These experiments demonstrate that the synthetase and the ribosome compete for binding to the wild-type thrS leader mRNA. The presence of an approximatively 10-fold excess of tRNAThr over synthetase completely relieves the inhibitory effect of the synthetase on ribosome binding. This effect is specific, since tRNAPhe has no effect (175). This shows that the tRNAThr acts as an antirepressor, suggesting that the intracellular concentration of uncharged tRNA (aminoacylated tRNA being trapped by elongation factor Tu) modulates the repressor activity of threonyl-tRNA synthetase. The repression-derepression double control provides a mechanism that allows precise adjustment to the rate of synthesis of ThrRS in response to intracellular fluctuations of both tRNAThr and synthetase concentrations to avoid the accumulation of uncharged tRNA in the cell.
Other Possible Regulatory Mechanisms.
Lysyl-tRNA synthetases: diversity and coordination. Lysyl- tRNA synthetases are synthesized from two distinct genes in E. coli: lysS and lysU (61, 269). Characterization of their structural genes has shown that the derived amino acid sequences are 86% identical (39, 155). The major species of LysRS (57.6 kDa) is encoded by lysS, located at 62 min on the chromosome (61), and the minor species (57.8 kDa) is encoded by lysU, located at 94 min (269). lysS is in the same operon as the prfB gene, which encodes peptide chain release factor RF2 (132), and is expressed constitutively (107, 108). lysU, on the other hand, is normally silent but is induced by various stimuli (106, 154) such as temperature shift, external pH change, anaerobiosis, the stationary phase, or the presence of specific metabolites including l-leucine in the growth medium (25, 107). A strain carrying a null mutation in lysU exhibited no noticeable phenotype. On the other hand, the disruption of lysS confers cold-sensitive growth on the cells, since lysU is particularly induced at high temperature. The expression of lysU is controlled by several genes such as lrp (190), hptR (189), rlu (108), and hns (123), which code for the leucine response protein, the heat shock sigma factor, a less well characterized regulatory locus, and a histone-like protein (H-NS) implicated in thermoregulation of lysU, respectively. Lrp, the leucine response regulatory protein, is a bifunctional transcriptional regulator which acts as a repressor or activator for several genes (190). It was shown genetically that lysU is part of the leucine regulon (122). The direct involvement of lrp in lysU expression was shown by DNase I footprinting; Lrp protein binds to DNA upstream of lysU (70, 157). lysU, normally repressed by the Lrp protein, is derepressed upon addition of l-leucine. The demonstration that lysU is part of the leucine operon raises the question of how the increase in lysU activity functions and how it interacts with the global response of the lrp regulon.
Heat shock activation (189) is distinct from the lrp-mediated regulation of lysU by metabolites. The role of hptR in thermoregulation of lysU is unclear, since the induction of hptR, which controls sigma-32, without temperature shift does not cause lysU derepression (123, 268). The expression of lysU is also under the control of a locus named rlu, located at 49.5 min on the E. coli map (108). In addition to the transcriptional control of lysU, a small region immediately downstream of the initiation codon of the lysU gene is required for high levels of expression. This sequence is similar to that of the downstream box, a potential translational enhancer sequence complementary to nucleotides (1469 to 1483) of 16S rRNA that activates translation of highly expressed genes such as ribosomal protein genes. This element may play a role in the thermoregulation of lysU (122).
The cadABC operon maps upstream of lysU. cadA encodes lysine decarboxylase, which produces cadaverine from lysine; cadaverine inhibits lysine tRNA synthetase activity. cadA expression, like that of lysU, is stimulated by anaerobiosis, acidic external pH, and H-NS (249). Common regulatory elements might be shared by cadABC and lysU. This situation suggests that the mechanism responsible for the coordinate expression of lysU has other genes outside the leucine lrp regulon. Under harsh conditions such as low pH and anaerobiosis, the coordinate induction of lysU and cadA can be explained by the fact that cadaverine is an inhibitor of LysRS. LysRS, encoded by lysU, is more resistant to inhibition by cadaverine than is the synthetase coded by lysS (21a).
The last few years have seen considerable progress in understanding the complex regulation of LysRS. It is now considered likely that the inducible LysRS is coordinately regulated with several other regulatory pathways in response to various environmental conditions.
Methionyl-tRNA synthetase. Methionyl-tRNA synthetase in its native form is a dimer of one type of subunit (76 kDa). It is coded for by the metG gene. The DNA sequence of the entire gene has been determined (10, 50). As discussed earlier, physiological studies showed that MetRS synthetase synthesis is regulated by the aminoacylation of tRNAMet (34). The DNA sequence upstream from metG indicates the presence of an open reading frame which is transcribed divergently from metG. This open reading frame codes for a methionine-rich protein of 39.9 kDa (50, 51) called mrp (metG-related protein). The metG gene is expressed from two promoters: the downstream promoter is located in the mrp-metG intergenic region, and the upstream promoter is located within mrp. mrp is expressed from a promoter which also lies within the intergenic mrp-metG region, so the –35 regions of the mrp and metG proximal promoters overlap. In addition, a terminator is located at the beginning of the mrp open reading frame between the two metG promoters. S1 nuclease mapping clearly indicates that transcription of metG starting at the upstream promoter is attenuated at this terminator. The metG leader shows no open reading frame but may form a tRNA-like secondary structure with a CAU anticodon-like sequence. An autoregulatory model in which an excess of methionyl-tRNA synthetase binds to this leader and somehow affects transcription termination at the terminator has been proposed. Further experiments are needed to prove the proposed model (51).
As discussed above, great diversity is observed in E. coli gene organization and control mechanisms of aminoacyl-tRNA synthetases. Recent progress has now allowed a comparison between gram-positive and gram-negative organisms which can provide valuable information on how bacteria regulate and coordinate the expression of the synthetases.
Aminoacyl-tRNA synthetases specific for 11 amino acids have now been cloned from different Bacillus species and sequenced (220). There are no significant structural differences between the proteins of E. coli and Bacillus aminoacyl-tRNA synthetases. However, the following differences in gene organization and expression between Bacillus subtilis and E. coli aminoacyl-tRNA synthetases are significant.
(i) The chromosomal locations and arrangements of the B. subtilis genes are completely different from those of the gram-negative counterpart.
(ii) In contrast to E. coli, gram-positive organisms lack an enzyme capable of charging a tRNA with glutamine (282). In B. subtilis, a single glutamyl-tRNA synthetase attaches glutamate to both tRNAGlu and tRNAGln. The mischarged Glu-tRNAGln is transformed into Gln-tRNAGln by a specific aminotransferase (282, 283). The gltX gene (encoding the synthetase) seems to be expressed in a constitutive manner.
(iii) In B. subtilis are at least two examples of the existence of multiple genes for an amino acid-tRNA synthetase of the same specificity (for threonine, thrS and thrZ; for tyrosine, tyrS and tyrZ) (72, 102, 217).
(iv) Although in E. coli the mechanisms of regulation of amino acid-tRNA synthetase gene expression are different, in B. subtilis most of the genes isolated appear to be regulated by a common mechanism: transcriptional antitermination.
Strongly conserved structural regulatory elements occur in the untranslated leader regions of most of the gram-positive amino acid-tRNA synthetase genes and of several amino acid biosynthetic operons (72, 89, 102, 218, 219). Each of the leader regions is about 300 bases long (see Fig. 4A for that of thrS) and has several conserved elements, most notably an 18-nucleotide sequence, known as the T box, immediately upstream of a factor-independent terminator (90, 101, 102, 218). All these genes are highly and specifically induced by starvation for their cognate amino acid but not by general amino acid starvation (101, 220, 221). The threonyl-tRNA synthetase genes, thrS and thrZ, are, in addition, autorepressed by overproduction of the synthetase itself (71, 218). The response to starvation appears to be mediated by the interaction of the cognate uncharged tRNA with the mRNA leader region, which promotes formation of an antiterminator structure.
Two sites of interaction between the uncharged tRNA and the leader have been proposed. The first is between the anticodon loop of the uncharged tRNA and a "specifier codon" that is postulated to be a bulged loop on the side of a stem-loop structure located at the beginning of every leader RNA (Fig. 4B). Changing the specifier codon is thought to affect the specificity of the interaction of the tRNA with the leader mRNA (88, 221). The second proposed interaction between the uncharged tRNA and the leader occurs between the CCA-3' acceptor end of the tRNA and a complementary triplet in the T box of the leader mRNA. It is this interaction that is believed to stabilize the antiterminator stem-loop (90, 221). The mechanism of autoregulation of the thrS gene probably involves leader determinants the same as or similar to those involved in induction by amino acid starvation. However, although the codon-anticodon interaction and the interaction between the CCA-3' end of the tRNA and the T box are important for both induction and autoregulation of the aminoacyl-tRNA synthetase genes, they are not always sufficient (221).
In addition, the expression of the threonyl-tRNA synthetase gene is regulated in parallel to the growth rate. This regulation also occurs at the level of antitermination but is independent of the specifier codon and may represent a second overlapping mechanism of controlling genes of this family. Although this type of regulatory mechanism has not been found in E. coli, it would not be surprising to me if it were identified among the genes left to be characterized.
The aminoacyl-tRNA synthetases each catalyze essentially the same reaction in protein synthesis. Surprisingly, they are very different in size, subunit structure, and primary sequence. They all appear to be under metabolic control. In half of the aminoacyl-tRNA synthetases, an individual regulatory response to limiting amounts of the cognate amino acid is superimposed on metabolic regulation. This individual response is not always easy to observe, because adequate bradytrophs are not always available. Therefore, it is quite possible that eventually most, if not all, of the synthetase genes will be shown to be regulated this way. It is possible that the two phenomena, i.e., metabolic regulation and derepression upon starvation, evolved to solve two different problems. Metabolic regulation would result in a steady-state level of aminoacyl-tRNA synthetase, whereas derepression upon starvation would immediately, and sometimes transiently, adjust the level of the synthetase, thereby avoiding large changes in acylated-tRNA levels. However, instead of being superimposed upon metabolic regulation, the individual regulatory responses to amino acids may be responsible for the metabolic effects on gene expression. There is, however, no experimental evidence for this hypothesis. What is even more surprising is that the mechanism of derepression upon amino acid starvation appears to be different in E. coli for each of the aminoacyl-tRNA synthetases for which a mechanism can be proposed on the basis of experimental evidence. Although expression of alanyl-tRNA synthetase is controlled in vitro by the free amino acid or the aminoacyl-adenylate, expression of phenylalanyl-tRNA synthetase appears to be controlled by tRNA aminoacylation through an attenuation mechanism. The molecular mechanism of thrS autoregulation is now well documented in vivo and in vitro; the synthetase binds to its mRNA in front of its structural gene, thereby inhibiting translation of the gene. Under threonine starvation conditions, the derepression described above could be a consequence of the same mechanism via the competition between uncharged tRNA and mRNA for the binding of synthetase. The competition is explained by the fact that the operator site of the thrS mRNA, where the synthetase binds, shares homology with the cognate tRNAs. Thus, both pheST and thrS have their respective tRNAs as effectors. In gram-positive bacteria, the respective tRNAs are also effectors for the expression of most of the aminoacyl-tRNA synthetases by favoring transcriptional antitermination. However, via different mechanisms, tRNA seems to be the effector most commonly used to modulate aminoacyl-tRNA expression.
The expression of pheST is regulated over a 20-fold range, as shown by the attenuator mutations. The translational operator mutants of thrS show that this gene is also regulated over a 10-fold range. Thus, the magnitude of the change in expression is not very different, whatever the mechanism. This brings us to several questions. Is the elaboration of different regulatory mechanisms purely accidental, or is there a profound reason for the different mechanisms? Have these mechanisms evolved in order to tie the regulation of aminoacyl-tRNA synthetase gene expression to the overall physiology of the cell?
Aminoacyl-tRNA synthetases might have appeared relatively early in evolution. In the earliest forms of life, there may have been self-replicating structures of nucleic acid and, soon thereafter, proteins. The earliest proteins which facilitated the biosynthesis of protein may have been the aminoacyl-tRNA synthetases. Autogenous regulation might be one of the first mechanisms governing specific gene regulation, whereas attenuation could have evolved afterward. It will be important to know whether the different aminoacyl-tRNA synthetases are derived from different progenitors. Their different mechanisms of regulation, as well as their sequence differences, suggest that they have evolved separately. Studies of the expression of aminoacyl-tRNA synthetase genes in different organisms, such as archaebacteria, might shed some light on this subject.
Studies of the regulation of expression of aminoacyl-tRNA synthetase genes at the molecular level appear to be a very promising technique for generating knowledge of the general mechanisms of recognition of nucleic acids by proteins.
Introduction.
Initiation complex formation on 70S ribosomes in E. coli involves, in addition to initiator fMet-tRNAfMet, mRNA, and GTP, the cooperative interaction of three proteins, initiation factors IF1, IF2, and IF3 (85, 105). Until now, no convincing data have indicated the existence of additional factors. Numerous in vitro experiments demonstrate that IF1 and IF3 shift the equilibrium from 70S ribosomes toward dissociation into 30S and 50S subunits. Under physiological conditions, the ribosomal subunit equilibrium is toward 70S formation, and IF1 serves to increase the rate constant for both the dissociation and the association of the 70S ribosome, whereas IF3 shifts the equilibrium strongly toward dissociation by binding to the 30S particle (73). IF3 selects the initiator tRNA in the presence of the initiation codon triplet as well as on a natural mRNA template (see below) (96, 98). IF3 action distinguishes the initiator tRNA from elongator tRNAs by specific domains in the tRNA and not by its formylmethionine. Part of the information is located in the anticodon stem-and-loop region (96). IF3 selectively destabilizes polynucleotide-primed 30S complexes formed with elongator tRNAs (98, 208, 232). The third initiation factor, IF2, directs fMet-tRNAfMet binding to the 30S subunit. This initiation factor might also exclude noninitiator tRNAs from the P site while allowing formylated, charged initiator tRNA to enter. This reaction is stimulated by IF1 and GTP. In addition, IF2 helps the association of the two subunits and has a GTPase activity in the presence of ribosomes (for reviews, see references 84, 86, 91, and 104).
In the absence of tRNA or initiation factors, the 30S particle by itself binds the proper initiation region on mRNAs (97). The sequence of events directed by initiation factors is summarized in Fig. 5: IF1 and IF3 are released before or during the joining of the subunits, whereas IF2 is released after their joining and requires GTP hydrolysis. The mechanism of formation of the initiation complex and the role of factors are discussed in detail in chapter 59 of this volume. The three factors are present in cells at high enough concentrations to saturate the free 30S particle (110). The steady-state levels of initiation factors are coordinately regulated in exponentially growing bacteria relative to one another and to ribosome levels. All levels rise as a function of increasing growth rate (110), suggesting that the cellular levels of initiation factors are under metabolic control. Neither IF2 synthesis nor IF3 synthesis is under stringent regulation in vivo (58, 225). However, in apparent contradiction to the in vivo results, the synthesis of IF3 in a coupled transcription-translation system in vitro is inhibited in the same concentration range of ppGpp as was reported to affect synthesis of ribosomal proteins (58, 152).
All of the initiation factor genes have been cloned and mapped: infA for IF1, infB for IF2, and infC for IF3. They are dispersed on the bacterial chromosome and, with the exception of infA, are associated with other components of the protein biosynthetic apparatus (Fig. 6) (threonyl- and phenylalanyl-tRNA synthetases and ribosomal proteins L20 and L35 for IF3; tRNAfMet, NusA, and ribosomal protein S15 for IF2).
Initiation Factor IF1.
Of the functions of the three factors, that of IF1 is the least clear, since no specific role has been assigned to this protein. IF1 is the smallest of the initiation factors (M w = 8,100), consisting of 71 amino acid residues (209). The gene for IF1, infA, has been cloned, sequenced, and mapped to about 20 min on the E. coli chromosome (242). The infA gene is transcribed from two promoters to yield two monocistronic mRNAs, both ending at the same terminator. Neither transcription nor translation of infA is affected by high cellular levels of IF1. The promoter resulting in the shorter transcript increases in activity as a function of growth rate (47). Homologous genes have been identified in B. subtilis (18) and in the chloroplasts of several plants (195). The high degree of homology shows that IF1 is a conserved protein and suggests that it plays an important role in the cell. Until recently, all data came exclusively from a number of in vitro assays of 70S ribosome dissociation and association. Alone, IF1 binds poorly to 30S, but upon addition of IF2, the affinities of both factors are increased. IF1 stimulates the binding of fMet-tRNA to 30S or 70S ribosomes and stimulates β-galactosidase synthesis in a DNA-linked highly purified transcription-translation system eightfold (138).
To elucidate the function of IF1 and to determine whether the protein is essential to cell growth, the chromosomal copy of infA was disrupted (46). Cell viability is maintained only when infA is expressed in trans with a plasmid whose replication is dependent on the thermosensitive polA gene. Replication can be blocked at the nonpermissive temperature (42°C), thereby leading to the loss of infA. As IF1 levels fall, polysomes become smaller and cell growth decreases. Thus, IF1 is an essential gene. The fact that cells depleted of IF1 exhibit few polysomes suggests that IF1 indeed functions in the initiation phase of protein synthesis.
Initiation Factor IF3.
IF3 protein has an M w of 20,564, contains 180 amino acids, and is composed of two domains of approximately the same size that are separated by a flexible linker (66). An E. coli strain with a thermosensitive IF3 was isolated by an in vitro assay procedure from a collection of nitrosoguanidine mutants enriched for thermosensitivity by the tritiated amino acid suicide method (256). The isolation of a lambda transducing phage complementing the IF3 mutant permitted the localization of infC at 37.4 min on the E. coli chromosome (60, 257). This region carries seven other genes (Fig. 7) in the following order: thrS infC rpmI rplT pheS pheT himA. These genes code for threonyl-tRNA synthetase, IF3, ribosomal proteins L35 and L20, the small and large subunits of phenylalanyl-tRNA synthetase, and a subunit of the host integration factor that is required for the integration of phage λ DNA into the host chromosome by site-specific recombination, respectively. All of these genes are transcribed in the same direction, i.e., counterclockwise from thrS to himA (260).
Transcriptional units. The DNA fragment starting at thrS and ending with himA has been entirely sequenced (160, 165, 239). The structural genes thrS and infC are separated by only three nucleotides (150). Thus, thrS transcripts must continue into infC. The amounts and the origins of mRNA transcripts that penetrate into the IF3 gene were measured in vivo and in vitro. The results of these measurements show that infC is cotranscribed with thrS (26). The results also indicate that there is approximately five times more infC mRNA than thrS mRNA. This value is in agreement with data showing that there is approximately five times more IF3 protein (110, 111) than threonyl-tRNA synthetase (187) in the cell.
In addition to the thrS promoter (pthrS), which generates thrS-infC cotranscripts, two promoters within the thrS structural gene have been identified by in vivo S1 mapping experiments (26). The first, called p 0 , is situated in the middle of the thrS gene, between positions 1189 and 1219 of the 1,926-bp-long thrS coding region, and is capable of synthesizing transcripts which enter infC. Another promoter (p 0' ) is found between bp 1716 and 1746. The relative intensities of the S1 nuclease-protected fragments and the quantitative mRNA hybridization measurements indicate that the promoter closest to the infC structural gene (p 0' ) is the major site at which infC transcripts are initiated (26) (Fig. 7).
The infC coding region itself carries a promoter, p 1, which has been shown in vitro to direct transcription toward the rplT gene, located 347 nucleotides downstream from the end of infC (65, 87). The terminator (t 1), situated downstream of infC, is not very efficient: 80% of the in vitro transcripts originating at p 1 read through t 1 and extend into the L20 structural gene. The p 1 promoter within infC is weaker than p 0' . Thus, the p 0' promoter is the main promoter for the expression of the infC, rpmI, and rplT region of the chromosome. Polycistronic mRNAs that contain thrS, infC, and rplT may also exist. Thus, it seems that initiation and termination signals are arranged to yield a set of overlapping transcripts from this region of the chromosome.
Control of infC expression. Experiments in a coupled transcription-translation system in vitro do not indicate a specific inhibition of IF3 synthesis after the addition of up to a fivefold molar excess of IF3 over ribosomes. IF3 synthesis was inhibited by the addition of IF3 at higher concentrations, but the effect did not appear to be specific (152). However, the nonspecific inhibition may have masked a more specific effect of its expression in a crude in vitro system. The results of in vivo experiments on the control of infC expression clearly show that cellular levels of IF3 regulate the expression of infC at the translational level (26). This conclusion is based on several lines of experimental evidence. First, an infC mutant which overproduces an altered form of IF3 does not overproduce infC mRNA. Second, infC-lacZ protein fusions, but not infC-lacZ operon fusions, are derepressed in trans by two infC mutant alleles. Third, plasmids which overproduce IF3 cause the repression in trans of infC-lacZ protein fusions but not of infC-lacZ operon fusions. Fourth, conditions which cause the repression or derepression of hybrid β-galactosidase synthesis from an infC-lacZ protein fusion have no effect on hybrid infC-lacZ mRNA synthesis from the same gene fusion. These results show that changes in IF3 levels cause corresponding changes in the rate of synthesis of IF3 but not at the transcriptional level. Therefore, IF3 must negatively affect the translation of infC mRNA in vivo. S1 mapping and quantitative mRNA hybridization experiments show that infC expression is controlled independently of thrS expression.
The nucleotide sequence of the translational initiation region of the infC gene breaks many rules followed by most other E. coli mRNAs (74, 75, 76). The initiation codon AUU is unique among E. coli mRNAs. The existence of a second potential ribosome binding site, AGGU between the probable Shine-Dalgarno region and the initiator AUU codon, is also very rare. Finally, downstream of the initiation codon is a GC-rich sequence which is very unusual in ribosome binding sites. Yet the IF3 message is translated at a high rate in vitro (152). These unusual features of the translation initiation region of infC led Gold et al. (76) to postulate that the translation of infC mRNA may involve a mechanism different from that used by other E. coli mRNAs.
The peculiarity of the E. coli infC gene in starting with an AUU initiation codon (207, 239) also extends to other bacterial IF3 genes which have been sequenced (112, 158, 207). Alteration of the AUU translation start to AUG abolishes control (28), thus clearly showing that the abnormal initiation triplet is necessary for regulation. It also appears that alteration of the normal AUG initiation codon of several genes to AUU is sufficient to make these genes regulatable by IF3 levels in the cell (J. Sussman, J. Chang, and R. Simons, personal communication; K. Engst, C. Sacerdot, and M. Springer, unpublished data). IF3 does not specifically recognize AUU. Other abnormal initiation codons which can differ from AUU at every position also become regulated by IF3 (J. Sussman, J. Chang, R. Simons, C. Sacerdot, and M. Springer, personal communication). It is thus likely that IF3 regulates its expression in vivo by binding to the ribosome rather than the mRNA and that, once on the ribosome, it inspects the triplet anticodon of the initiator tRNA and the initiation triplet of the mRNA.
Thus, IF3 is able to differentiate between normal and abnormal initiation codons. This is exactly the role for IF3 proposed earlier on the basis of in vitro experiments. IF3 stimulates translation initiation with the AUG initiation codon and inhibits initiation with some other codons, in particular AUU (13, 145). It was shown in vitro, using toeprint technology (99), that IF3 selects initiator tRNAs over elongator tRNAs in response to a codon adjacent to or overlapping the true initiation codon (96, 98). The anticodon stem and loop of the initiator tRNA together with the initiation codon of the mRNA can be thought of as a unit. The strength of the interaction between the anticodon of initiator tRNA and the start codon in mRNA presumably plays a role in the selection of the initiation codon. This selectivity is found with AUG, GUG, and UUG initiation codons but not with AUU. The guanosine in position 3 of the initiation codon is probably essential for IF3 selection of initiator tRNA. AUU gives poor toeprints with initiator tRNA fragments which have been destabilized by IF3. The exclusion of initiator tRNA from an AUU translation start provides a mechanism for autogenous translational repression of IF3 expression. When the 30S subunit-IF3 complex binds to an mRNA with a standard initiation codon, IF3 accelerates the rate at which the preternary complex changes to a productive ternary complex (96). If the 30S-IF3 complex binds to an mRNA with an abnormal initiation codon, IF3 instead accelerates dissociation of the preternary complex (98, 208, 232). It is in this way that autoregulation by IF3 is likely to be related to IF3 enzymatic function.
Is the infC gene essential? A few thermosensitive mutants mapping in infC have been isolated by P1-mediated localized mutagenesis (M. Springer, personal communication). These mutants, however, are thermosensitive in minimal medium but not in rich medium. The thermosensitivity phenotype does not seem to be reversed by the addition of a single metabolite to the growth medium. This special phenotype means either that IF3 is not absolutely essential in rich medium or, more likely, that the mutant is leaky enough to permit growth in such a medium. To definitively answer the question, a gene disruption which does not affect the expression of the adjacent essential genes is necessary.
Initiation Factor IF2.
Initiation factor IF2 is the largest of the initiation factors and is present in bacterial cells in two forms, IF2α and IF2β. The two proteins, with M w 97,300 and M w 79,700, respectively, are encoded by the same gene, infB (53, 201, 238). The infB gene is located in a complex operon at 68.5 min on the E. coli genetic map near argG (Fig. 6 and 8). This operon (139, 201) also contains the genes metY, coding for a minor form of the initiator tRNAMetf2; nusA, coding for a protein involved in transcription termination and antitermination; p15A and p15B, coding for two proteins of unknown function and with molecular masses of 15 kDa, as determined by sequence analysis (p15A has an apparent mobility on sodium dodecyl sulfate [SDS]-polyacrylamide gels of 21,000); and p35, with a molecular mass of 35,000. The metY gene (117, 118) differs at nucleotide 47 from metZ, which is located at 61 min, and codes for the major form of the initiator tRNA (55).
The genes for ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) are located further downstream from infB (210, 211, 212, 231), as are the deaD gene, coding for a putative DEAD box RNA helicase, and mtr, coding for a tryptophan permease (100, 264).
Organization of the infB operon. The entire DNA fragment starting from metY and ending after the mtr gene has been sequenced (120, 121, 212, 229, 231, 238, 241, 243). This establishes the order of the genes as follows: metY p15A nusA infB p15B p35 rpsO pnp deaD mtr (Fig. 8). Maxicell analysis showed a relatively strong expression of p15A and p15B, whereas p35 is synthesized more weakly. p15B or truncated p35 might be necessary for maximum cell growth (42). The direction of transcription is counterclockwise from metY to pnp. The reading frames for nusA and infB are the same, and the two genes are separated by 21 bp (between the second stop codon of nusA and the AUG start codon of infB). It is quite remarkable that this complex operon has a very similar structure in the gram-positive bacterium B. subtilis, which separated from E. coli early in evolution. The conservation of the three proteins p15A, p15B, and p35 suggests that they have a functional role in vivo (248).
Several transcriptional start and termination sites were identified between metY and pnp. Two functional promoters were found upstream of metY. Transcripts initiated at p 1 begin 7 nucleotides upstream of metY, while p –1 initiates transcription 86 nucleotides upstream of the initiator tRNA gene in an AT-rich region which might be involved in the control of the operon (80, 227, 228). A third promoter, p 2, is located between metY and p15A. Ninety percent of transcription initiated at p –1 and p 1 terminates at the two terminators t 1 and t 2, located between metY and p15A, to give short initiator tRNA precursors.
The processing of initiator tRNA from the p –1 and p 1 readthrough transcripts includes an RNase III cleavage which separates a short tRNA precursor from the downstream p15A nusA infB polycistronic mRNA (230). Possible minor promoter activities have been detected upstream from nusA and infB when the genes are cloned in recombinant vectors, but it is uncertain whether these activities play a role in vivo (205). S1 mapping did not detect promoters for p15B and p35, which are presumably cotranscribed with nusA and infB. The demonstration of readthrough downstream of infB into p15B and the identification of a transcript containing nusA, infB, p15B, and p35 support this conclusion (243). The absence of Rho-independent transcription terminators between p35 and rpsO suggests that transcription starting upstream of metY can continue as far as rpsO. The presence of strong promoters preceding rpsO and pnp was confirmed by S1 mapping, which also demonstrated a weak terminator after rpsO and several RNA processing sites: an RNase III site between rpsO and pnp, and RNase E sites both upstream and downstream of the rpsO transcriptional terminator (230) and in pnp (92, 230) (Fig. 8). Therefore, expression of rpsO and pnp is not strongly linked to the upstream genes. However, the results from S1 experiments also suggest that RNA polymerase expresses rpsO and pnp very weakly by readthrough from upstream promoters (P. Regnier, unpublished data).
In vivo analysis of the nusA-infB operon has also been performed by deletion mapping. Proteins synthesized by deletion plasmids were examined in a minicell system (119, 181). Eliminating the –35 region upstream of metY affected the synthesis of p15A, NusA, IF2α, IF2β, and p15B but did not affect the synthesis of S15 or polynucleotide phosphorylase. This finding, however, does not necessarily contradict the S1 mapping data, since the contribution of transcription originating upstream of rpsO may be small enough relative to that originating from p 1 and p 2 that its absence is difficult to detect. These findings demonstrate that rpsO and pnp are mainly expressed independently of the metY-p15A-nusA-infB-p15B operon.
Two translational initiation sites are used to express initiation factors IF2α and IF2β. The infB gene, subcloned as a 3-kb fragment, expresses both IF2α (97.3 kDa) and IF2β (79.7 kDa) (205), indicating that both IF2 forms originate from the same gene. N-terminal amino acid determination of IF2α and IF2β indicates that IF2β starts 471 bp downstream from the IF2α start codon (240). The AUG initiation codon for IF2α is preceded by a strong Shine-Dalgarno sequence, whereas the internal GUG start codon for IF2β is preceded by a purine-rich sequence which is a rather weak ribosome binding site. Immunoblot analysis of cells rapidly lysed in SDS buffer consistently revealed IF2α and IF2β at a molar ratio of 2:1 (109). Moreover, two forms of IF2 have been found in every species of enterobacterium examined thus far and in gram-positive bacteria as well (111, 113). These results suggest that the two forms are actually present in bacteria and that IF2β is not the result of proteolytic cleavage of IF2α during isolation.
A number of other experiments indicate that IF2β results from independent translational initiation rather than from a precise proteolytic cleavage of IF2α (199). A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids of β-galactosidase. The fused gene expresses two products (170 and 150 kDa) that correspond to the fused proteins IF2α–β-galactosidase and IF2β–β-galactosidase, respectively. This confirms that the two IF2 forms differ at their N termini in vivo. A deletion of the 5' nontranslated region of the fused gene, including the ribosome binding site of IF2α, results in loss of expression of the IF2α–β-galactosidase fusion protein but not the IF2β fusion protein. This is consistent with two initiation sites.
Further evidence for the initiation of protein synthesis at the putative IF2α and IF2β start sites comes from the use of an in vitro dipeptide synthesis assay (36). This coupled transcription-translation system is dependent on exogenous DNA and synthesizes formylmethionyl dipeptides in response to the first two codons of translated open reading frames. The in vitro synthesis of dipeptides faithfully mimics in vivo gene expression. A DNA fragment containing the entire infB gene was cloned into a plasmid vector, and the resulting recombinant DNA was used as a template in assays containing fMet-tRNA and various labeled aminoacyl-tRNAs. Synthesis of both formylmethionyl-threonine (corresponding to the IF2α start sequence) and formylmethionyl-serine (corresponding to the IF2β start sequence) was detected (177).
Functions of the two forms of IF2. As an approach to elucidating the function of the N-terminal part of IF2 and more specifically why two forms of this factor coexist in the cell, mutations that allow the expression of just one form of IF2 to the exclusion of the other were constructed (240). When deletions in the N-terminal region of infB were used, obtaining mutant plasmids in which only IF2β was expressed was easy. The construction of mutations inactivating IF2β expression proved to be more complicated and in fact revealed a second translation initiation codon for IF2β (240). When the GUG initiation codon of IF2β was mutated, IF2β synthesis was reduced by the mutations but was not abolished. The residual IF2β results from translational initiation at an AUG codon located 21 bases downstream from the mutated GUG. Determination of the N-terminal amino acid sequences of the two forms identified the two internal start sites. Only by mutating both the GUG and the AUG start codons for IF2β was it possible to construct plasmids expressing just IF2α. By using a strain carrying a null mutation of the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, it was possible to test the function of the different IF2 proteins carried on plasmids (140, 240). As had already been strongly suggested (42), the infB gene was shown to be essential. In addition, each of the two forms of IF2 can support growth of E. coli at near-physiological levels, but growth is retarded at 37°C relative to that when the two forms are present. Moreover, if only IF2β is present, the cells are cold sensitive. It was also observed that when a truncated version of infB expressing only the C-terminal 55 kDa of the protein was overproduced, it was also capable of supporting growth of E. coli (140).
In conclusion, since both forms of IF2 are required for maximum growth of the cell and since the two forms have been conserved through evolution, they may have acquired some specialized though not essential function (250).
Amino acid sequence of IF2. Analysis of the amino acid sequence of IF2 (238) reveals some remarkable features. The N-terminal part of the IF2α primary sequence has two adjacent regions that are rich in alanine and charged amino acids and show striking periodicities in their sequence. Near the center of IF2 is a region with an approximate M w of 17,000 that is characteristic of a GTP binding domain. This motif was originally identified in a small mammalian protein of the ras oncogene family (P21) and in the related yeast ras protein and has been called the G domain (159). Structural and functional analyses of the different regions of IF2 showed that the catalytic properties of IF2 are localized in the C-terminal half of the protein and include the G domain (140).
Regulation of expression of the nusA- infB operon. Because NusA affects transcription, it is pertinent to ask whether NusA affects the expression of its own operon. The NusA protein seems to be pleiotropic in the domain of transcriptional termination, and its precise role in vivo has yet to be determined (184). The gene was first identified by a mutation, nusA1, which prevented the growth of wild-type λ bacteriophage at high temperature without having a significant effect on bacterial physiology (68). The nusA1 mutation allows growth of N-independent variants, such as λ nin. Because the λ N protein is required to overcome transcriptional termination in the nin region (deleted in the λ nin variants), this phenotype implied that the wild-type NusA protein coupled with N acts as an antitermination factor. A number of studies demonstrate that NusA can also stimulate pausing and termination (62, 81, 119, 135, 277). A second mutant, nusA2(Ts) (ts1), with a temperature-sensitive defect in the structural gene for NusA, has also been isolated (182). Strains carrying this mutation grow at 32°C but fail to grow at 42°C. The efficiency of transcriptional termination in the nusA2(Ts) mutant at both Rho-dependent and Rho-independent terminator sites is abolished or markedly decreased. The results with nusA2(Ts) suggest that the function of NusA is to enhance termination of transcription (182). The same conclusion has been reached by analysis of amber mutants of nusA (185).
To test whether the expression of the nusA-infB operon is regulated autogenously by the nusA gene product, the effect of nusA mutations on the rate of synthesis of NusA and IF2 proteins was studied (183). The cellular levels of NusA, IF2α, and IF2β in the nusA2(Ts) mutant were about twofold higher than those in the wild type at 32°C and increased to three- to fourfold higher at 42°C. The expression of nusA and infB was also examined in a nusA amber mutant [nusA105(Am)] which carries a temperature-sensitive amber suppressor. The mutant grows normally at 32 but not 42°C. When a mutant culture was transferred from 32 to 42°C, the amount of NusA decreased to 30% of the level of the wild type upon inactivation of the suppressor. In contrast, the amount of IF2α and IF2β increased about fivefold at 42°C. These results are consistent if one assumes that the synthesis of IF2, like that of NusA, is negatively regulated by the nusA gene product.
Another approach to verifying the regulation model was to use gene fusions between nusA, infB, and lacZ (200). Protein and operon fusions coupling various regions of the nusA-infB operon to lacZ have been constructed on λ bacteriophages. Protein fusions between nusA (or infB) and lacZ carry the metY promoter, the two terminator sites, p15A, and either a part of nusA or all of nusA and a part of infB. An operon fusion joined the proximal part of the nusA-infB operon containing the metY promoter, the two terminators, and half of p15A to a complete lacZ with its own translational signals but missing its promoter. Overproduction of NusA from a multicopy plasmid reduces by about twofold the activity of β-galactosidase. Although the effect is small, it is reproducibly observed, and both the operon and the protein fusions respond in the same way to NusA. On the other hand, plasmids which overproduce IF2 but not NusA have no effect on β-galactosidase activity, confirming that it is NusA and not IF2 which regulates the expression of the operon. The effect of NusA is felt throughout the operon at least as far as the infB gene. The structure of the nusA-infB operon suggests at least one possible mechanism for the autoregulation. NusA could be regulating the amount of readthrough at terminators t 1 and t 2 after metY. However, NusA might also affect the expression of the operon at a site other than the t 1 and t 2 terminators (35).
Introduction.
Three peptide chain elongation factors, EF-Tu, EF-Ts, and EF-G, promote the binding of aminoacyl-tRNA, the exchange of guanine nucleotide bound to EF-Tu, and the translocation of peptidyl-tRNA and mRNA, respectively, on the ribosome (85, 104). Schematically, the elongation cycle can be summarized as follows. The first step is the formation of a ternary complex containing aminoacyl-tRNA, EF-Tu, and GTP (Fig. 9). The ternary complex binds to the ribosome–mRNA–peptidyl-tRNA complex, and GTP is hydrolyzed, allowing EF-Tu–GDP to dissociate from the ribosome. During the aminoacyl-tRNA binding reaction, a particular ternary complex is selected from the various species available on the basis of the codon-anticodon interaction between the ribosome-bound mRNA and the tRNA. EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu, thus allowing formation of the ternary complex. Peptide bonds are formed by transfer of the nascent peptide from its tRNA at the P site to the α-amino group of the newly bound aminoacyl-tRNA at the A site. The translocation reaction involves an EF-G–GTP-promoted movement of the peptidyl-tRNA from the A site to the P site with concomitant movement of the mRNA by three nucleotides. One molecule of GTP is hydrolyzed, and EF-G and GDP dissociate from the ribosome. The result is a ribosomal complex with a peptidyl-tRNA one amino acid longer, and the next mRNA codon becomes available for interaction with its cognate ternary complex.
Elongation Factor EF-G.
The EF-G gene (fus) was sequenced (288), and the three-dimensional structure of factor G from Thermus thermophilus and the crystal structure of EF-G complexed with GDP were determined at 2.85-Å (0.3-nm) resolution (1, 49). The gene is located at 73 min on the E. coli chromosome (Fig. 6; 10) in the so-called str region with three other genes. These genes code for ribosomal proteins S12 (rpsL) and S7 (rpsG), and one of the two genes codes for elongation factor EF-Tu (tufA); the genes occur in the order rpsL rpsG fus tufA. The four genes are under the control of a common promoter (127, 213) and are cotranscribed counterclockwise from rpsL to tufA. EF-G synthesis is regulated in vitro and in vivo by the translational repressor S7. The coordination of the synthesis of EF-G with that of ribosomal proteins is consistent with the fact that the cellular level of EF-G is approximately the same as that of ribosomes (170).
Elongation Factor EF-Tu.
Gene organization and structure. Two unlinked genes code for EF-Tu (16, 127). These genes, designated tufA and tufB, are remarkably similar in nucleotide sequence. Differences have been found at only 13 positions (3, 286). The corresponding gene products, EF-TuA and EF-TuB, are identical except for their C-terminal amino acid residues (5, 129). The tufA and tufB genes are located at two different positions on the linkage map of E. coli (127). tufA is located at 73 min in the str region with the genes coding for ribosomal proteins S12 and S7 and for elongation factor EF-G (see above) (Fig. 6). tufB is located at 88 min in the rif region (8). Four tRNA structural genes (thrU, tyrU, glyT, and thrT) code for 
, 
, 
, and 
, respectively, and immediately precede tufB (3).
Expression. The synthesis of EF-Tu is between 7- and 10-fold greater than that of ribosomal proteins and is not precisely coordinated with their synthesis (69, 196). At growth rates exceeding 1.0 doubling per h, the molar ratios of EF-Tu/ribosomes and EF-Tu/EF-Ts remain virtually constant. Above a rate of 1.0 doubling per h, the expression of tufA, tufB, tsf (coding for EF-Ts), and the ribosomal genes is thus regulated coordinately. At lower growth rates, this coordination persists for tufA and tufB, but the expression of tsf and the ribosomal protein drops faster than that of the tuf genes.
The fact that EF-Tu is encoded by two genes complicates the study of the regulation of its expression. A fruitful experimental approach to studying the expression of the two tuf genes has been the determination of intracellular levels of EF-TuA and EF-TuB in E. coli under various conditions of steady-state growth. Such determinations were made possible by point mutations which cause a change in the isoelectric point of one of the proteins, thereby making possible their separation by isoelectric focusing; their concentrations were determined by rocket immunoelectrophoresis (271, 273). These mutations do not alter the regulation of expression of tufA and tufB. Analysis of the intracellular tuf products revealed a constant ratio between the synthesis rates of EF-TuA and EF-TuB and between the intracellular amounts of these proteins at all growth rates studied (224, 272).
As discussed above, tufA is in the same operon as rpsL, rpsG, and fus and is expressed from a main promoter located upstream of rpsL. At least one-half of the cellular EF-Tu is derived from tufA (197, 273). However, tufA is expressed three times more than tufB, which means that the level of EF-TuA exceeds the levels of S7, S12, and EF-G expressed from the same operon. Evidence suggests the existence of a secondary promoter located in the coding region for EF-G which could be responsible for enhanced tufA expression (288, 290). The promoter is approximately 30% as active as the major promoter and might significantly contribute to the rate of EF-TuA synthesis (4, 271, 288, 289). Also, EF-Tu expression is not under the control of the translational repressor S7. This unequal expression of EF-TuA compared with that of S7, S12, and EF-G in addition to elevated mRNA transcription is due in part to its uncoupled translation.
The other locus on the genetic map of E. coli that codes for EF-Tu is the tufB gene. The four tRNA structural genes and tufB are included in the same transcriptional unit (Fig. 10) (115). The major promoter is located directly upstream of the thrU gene, but the existence of weak promoters located between the second and third tRNA genes and 32 bp upstream of tufB has been suggested (147). These results were obtained from analyses of gene fusions between different portions of the operon and different reporter genes (lacZ or the tetracycline resistance gene). This raises the possibility that differential expression of the genes in the tufB operon may occur in vivo.
Deletion of the four tRNA genes and their replacement by a single thrU-thrT hybrid gene only slightly affects the in vivo expression of tufB (172). Nor does the deletion affect the synthesis of β-galactosidase from a λ transducing phage lysogen carrying a tufB-lacZ protein fusion. Therefore, the tRNA genes are probably not essential for tufB expression (172). The thrU (tufB) operon of E. coli contains a cis-acting activation region, designated UAS for upstream activator sequence, upstream of the promoter. Deletion studies revealed that the high expression level of the operon requires this cis-acting sequence (275). Elimination of this sequence results in a 10- to 15-fold drop in transcription (270). A UAS has also been found upstream of the tRNA operons tyrT (141) and leuV (11) and of the p 1 promoter of the rRNA operon rrnB (77) (Fig. 10). Although the UAS of the stable RNA operons show a low degree of sequence homology (266), they have some common features. They are AT rich and display bending of the DNA helix (54, 77). Since the expression of these operons is highly coordinated in vivo, the question of whether a common activator protein binds to the UAS was raised. Early experiments indeed suggested that the sequence was the target of a trans-activating protein (276). Both in vitro and in vivo results suggest that this protein is Fis (factor for inversion stimulation) (17, 191, 192). The Fis protein is a heat-stable protein of 98 amino acids which is known to stimulate the inversion of various DNA segments by binding to cis-acting recombinational enhancers found in various prokaryotic DNA inversion systems (128, 130). Moreover, Fis induces bending of DNA, and the degree of bending influences the enhancer function of Fis (114). Transactivation of the thrU-tufB operon is drastically reduced in fis mutant cells (191).
It remains to be seen whether Fis by itself is sufficient for all of the transactivation or whether accessory proteins, in particular, EF-Tu, are involved. Earlier observations found that synthesis of RNA is stimulated by EF-Tu (265, 276). In addition to tufB operon control by activation, some results also suggest negative autoregulation of tufB expression (273).
The synthesis of EF-Tu, EF-Ts, and EF-G is also subject to stringent control in vivo. Their synthesis is reduced under conditions of amino acid deprivation or limitation of tRNA aminoacylation in relA + strains(15, 225). Furthermore, the synthesis of EF-TuB, directed by tufB DNA, in a cell-free coupled transcription-translation system (251) and the synthesis of its mRNA by purified RNA polymerase (171, 251) were specifically inhibited by ppGpp in vitro. This inhibition occurs at the level of transcriptional initiation (174). The concentration of ppGpp required to completely abolish synthesis of tufB mRNA is similar to the concentration of ppGpp which is normally observed in wild-type cells under conditions of amino acid starvation. Mizushima-Sugano and Kaziro (173) located the DNA sequence involved in the stringent control of the tufB operon by constructing various deletions, insertions, and point mutations of the promoter locus. The response of the mutants to ppGpp was examined in a cell-free transcription system by using purified RNA polymerase holoenzyme. The nucleotide sequence GCCC from positions –7 to –4 (with the mRNA initiation site designated position +1) is responsible for the selective inhibition of tufB transcription by ppGpp. The alteration of any nucleotide in the GCCC sequence leads to the loss of stringent control. This sequence is remarkably well conserved in many stringently controlled promoters (266).
Elongation Factor EF-Ts.
The gene for EF-Ts, tsf, found at 4 min on the E. coli genomic map, is part of an operon containing the ribosomal protein S2 (rpsB) (Fig. 6). The two genes are transcribed counterclockwise from rpsB to tsf (2). The single major promoter for the rpsB-tsf operon is located immediately upstream of rpsB. A structure similar to a Rho-independent terminator or attenuator occurs in the intergenic region between the two genes. This structure is preceded by a possible RNase III site (deduced by examination of the sequence). However, there is no evidence showing that this site is functional. A second strong transcriptional terminator lies distal to tsf at the end of the operon. As discussed above, EF-Ts synthesis is under stringent control and is coordinated with the synthesis of ribosomal proteins as a function of growth rate (170).
The process of peptide chain termination differs markedly from those of chain initiation and elongation, since the codon recognition molecules are proteins, not tRNA (21, 22, 31). Peptide chain termination is directed by one of three specific codons (UAA, UAG, and UGA, called ochre, amber, and opal, respectively) and results in release of the completed peptide from ribosome-bound peptidyl-tRNA.
The protein release factors are responsible for peptide chain termination. Two of the release factors, RF1 and RF2, have different codon specificities: RF1 recognizes UAA or UAG, and RF2 recognizes UAA or UGA. In agreement, pure homogenous RF1 and RF2 bind to ribosomes in the presence of their respective codons (247). The third factor, RF3, stimulates the release of polypeptide chains by RF1 or RF2; the action of this factor in vivo is largely restricted to UGA containing stop signals (82). RF3 binds GDP and GTP (32). The mechanism of stop signal recognition by the release factors, whether direct or indirect, is still poorly defined. The ribosomal peptidyltransferase is required for hydrolysis of peptidyl-tRNA upon chain termination. Perturbation of peptidyltransferase by the release factors leads to hydrolysis of the peptidyl-tRNA rather than to formation of peptide bonds.
Cloning of the Genes.
Recombinant plasmids prepared from the Clarke-Carbon colicin E1 bank (40) which carried the RF2 gene were initially identified by an immunoprecipitation assay (33) and their overproduction (about fivefold) of release factor RF2. On the other hand, a clone containing RF1 was identified by using the competitive relationship between a termination suppressor tRNA and release factors. It is known (12) that suppressor tRNAs which read termination codons compete with the corresponding release factor for the termination codon. A strain carrying a UAG mutation in lacZ and a glutamine-inserting UAG suppressor of low efficiency was used as a recipient for transformation by recombinant plasmids carrying chromosomal fragments. This method allowed (281) isolation of Lac– clones because of the overproduction of RF1 from the plasmid (237). A plasmid that overproduces RF2 which is analogous to that identified by Caskey et al. (33) was also found. The gene encoding RF1, designated prfA, is located at 26.7 min on the E. coli chromosome. The RF2 gene, designated prfB, is at 62.3 min (146), which is within the same operon as the lysyl-tRNA synthetase gene, as discussed above (132). Several mutants of RF1 and RF2 have been isolated, and they cause frequent misreading of stop codons or frameshifting as well as temperature-sensitive growth of the cells (59, 131). Hence, the reduced activities of release factors result in several translational errors in vivo, and these errors are likely to be caused by an abnormal pause of ribosomes at stop signals (233). In S. typhimurium, the supK gene encodes the Salmonella RF2 protein, and one mutation, supK584, carries an opal UGA mutation within the coding region. It has been proposed that the reduced intracellular level of RF2 causes autogenous suppression of the opal RF2 mutation (133). It is evident that changing either the concentration or the activity of the release factor leads to altered reading of the stop signal. Presumably, there is a complex kinetic balance between termination and competing processes, whether they be frameshifting, suppression, or specific amino acid incorporation at these sites.
After its initial characterization by stimulation of termination in vitro, RF3 received little attention until recently. Two groups (Grentzmann et al. [83] and Mikuni et al. [167]) identified the gene prfC and purified the protein (M w = 59,460). The protein displays homology to the family of G-binding proteins and in particular to factor EF-G. The gene maps at 99.2 min on the E. coli chromosome. To search for the gene encoding RF3, nonsense suppressor mutants were selected by random insertion mutagenesis on the assumption that a loss of function of RF3 would increase misreading of stop signals (83, 167). After mini Tn10 transposon mutagenesis, colonies were selected for simultaneous suppression of lacZ and leu UGA mutations (167) or enhanced suppression of a trpA UGA mutation in the presence of a weak UGA tRNA suppressor (83). The product of the gene binds GTP in vitro, as determined by UV cross-linking. It also markedly increases the formation of ribosomal termination complexes and stimulates fMet release in the termination assay in the presence of RF1 and RF2. The stimulation of fMet release is inhibited by GTP or GDP (at limiting trinucleotide concentrations). The proposed explanation is that the presence of GTP or GDP facilitates dissociation of the RF-terminator trinucleotide-ribosome complex, thus preventing formation of the termination intermediate. RF3 appears not to be essential for cell viability, but the mutant in which prfC is inactivated by a mini Tn10 insertion grows more slowly than wild-type cells (83).
Structures of the RF1 and RF2 Genes: Control of Their Expression.
The genes encoding RF1 and RF2 have been sequenced (116, 132, 146, 180). The sequence of RF2 was revised by Mikuni et al. (168). The molecular weights of RF1 and RF2 are 40,460 and 41,235, respectively. They are highly homologous in their primary structures, in accordance with structural similarities already suggested by antibody-binding studies (45, 223). The distribution of homologies suggests that the RF1 and RF2 release factors arose by gene duplication and subsequent evolutionary divergence.
The structural studies also suggest a very interesting new mechanism of translational regulation. An in-frame premature opal UGA termination codon is located within the RF2 coding region at codon 26. Comparison of the amino acid sequence with the nucleic acid sequence suggests that a +1 frameshift before the premature opal codon is required for complete translation of the RF2 mRNA (Fig. 11) (44, 45). This region of the protein was sequenced by Edman degradation to confirm the predicted reading frame. The sequence of the mRNA near the UGA codon has features consistent with the mechanism suggested. The in-frame UGA termination codon within RF2 is preceded by the leucine tRNA codon, CUU. The sequence CUU-UGA is rich in uracil and has been identified as a possible region of frameshift in different genes (7, 56, 67). Furthermore, tRNALeu has been reported to be a shifty tRNA with the ability to cause a 4-base translocation (280). The location of the opal codon within the early coding region of the peptide chain suggests that RF2 autoregulates its production by this in-frame UGA premature termination codon to limit its own translation. When RF2 is present in excess, peptide chain termination occurs at this site. When levels of RF2 are low, a readthrough frameshift mechanism permits translation of the full-length protein, since spontaneous termination in the absence of termination factors should not occur. Weiss et al. (279) demonstrated that an internal Shine-Dalgarno sequence immediately upstream of the UGA is necessary for frameshifting. This occurs via base pair formation with the 3' end of the 16S rRNA, which probably leads to ribosome stalling during elongation. The results of in vitro experiments using a coupled transcription-translation system are in accordance with the model of translational autoregulation: the addition of an RF2-overproducing plasmid inhibits the synthesis of RF2 (44). Evidence of in vivo negative-feedback regulation of RF2 was obtained by Curran and Yarus (48), who replaced the UGA stop codon by a UAG amber codon. Translation of the amber codon in a lacZ-prfB fusion using a suppressor tRNA competes with the frameshifting reaction, which proves one postulate of the translational autoregulation model. The RF2 frameshift mechanism has three essential features: (i) a pause or slow step in translation, (ii) a P-site tRNA that can pair stably to the message in alternative reading frames, and (iii) an upstream sequence that may activate and give orientation to the shift. This sequence is part of a Shine-Dalgarno sequence that may catalyze rephasing of the message by alignment of 16S rRNA.
The mechanism of such a translational autoregulation is quite exceptional. The other known examples of frameshifting are the bacteriophage T7 major capsid protein (56), a yeast mitochondrial cytochrome c oxidase gene (67), and the E. coli dnaX gene (267).
The genes for all of the protein factors known to be involved in peptide chain formation have been isolated. With the exception of the release factors, these protein factors are essential in vivo. The control of the expression of most of these factors has also been studied. The regulatory mechanisms operating on the genes of the soluble translational factors are only now being elucidated, and our understanding of them is less advanced than that of the genes for ribosomal components or aminoacyl-tRNA synthetases. In general, the genes for translational factors are not clustered together and are often found with genes whose products are involved in nucleic acid metabolism. Whether this arrangement is accidental, reflects the time of appearance of the soluble-factor genes, or has some role in coordinating expression of the translational and transcriptional apparatus is currently unknown. How the expression of protein factors relative to each other and to that of the other components of the translational apparatus is coordinated is still a mystery. The patterns of transcription, and in particular the presence of overlapping transcripts from multiple promoters and partially efficient terminators, indicate a potential for complex regulatory mechanisms. Cotranscription of initiation factor and elongation factor genes with genes such as metY and nusA (for infB), thrS (for infC), and rpsL and rpsG (for fus and tufA) might couple their synthesis to other components of the transcription-translation apparatus. On the other hand, the existence of internal promoters within these operons is probably a strategy for achieving differential expression of the genes under a variety of physiological conditions. Translational autoregulation is another way in which the expression of a given gene can be uncoupled from that of its cotranscribed neighboring genes.
In the case of initiation factor expression, these regulations do not appear to cover a large range. Overproducing NusA results in a maximum of fivefold repression of IF2. Any regulation implies, however, that controlling the concentration of soluble factors is important in one way or another for the cell. Such regulation might have a considerable effect on growth rate under certain conditions or might be of importance only on an evolutionary time scale. A new mechanism for negative autoregulation was shown in IF3 expression. IF3 in excess does not bind to mRNA or DNA but to the ribosome. Once on the ribosome, IF3 can differentiate between a normal and an abnormal initiation codon. This type of autoregulation, by which the repressor binds to the ribosome but not to nucleic acid, might not be restricted to IF3.
Termination of protein synthesis has traditionally been referred to as the hydrolysis of peptidyl-tRNA causing the release of the completed protein from the ribosome. However, as described above, a translational stop can be thought of in two ways: either as a complete stop mediated by the peptide-chain release factor or as a pause for more specialized events beyond the normal constraints of the genetic code. This pause might allow a diverse array of possible competing events, e.g., alternate readings of the genetic code, to occur at the stop signal. This raises two important questions: How are stop codons recognized as a signal for the termination of protein synthesis, and how is this recognition subverted to specify the alternative genetic codes? Many features of the termination mechanism, quite apart from the function of RF3, remain poorly understood. It has been proposed that the initial recognition of stop signals involves Watson-Crick base pairing with 16S RNA (180, 252). rRNA-mRNA base pairing also stimulates a programmed –1 ribosomal frameshift in decoding the E. coli dnaX gene (144). It is possible that release factors recognize an RNA-RNA duplex rather than single-stranded RNA. Obviously, further studies are necessary to uncover the molecular basis for stop codon recognition (a long-standing coding problem) and to decipher the alternate genetic code, the new frontier in translation coding.
I am most grateful to C. Condon, H. Putzer, M. Springer, R. H. Buckingham, and J. A. Plumbridge for their helpful comments and careful reading of the manuscript and to M. Springer for providing unpublished data.
This work was supported by the Centre National de la Recherche Scientifique (CNRS) (URA 1139), the Université Paris 7, the Ministère de l’Enseignement Supérieur et de la Recherche, EEC (contract CI1-CT90-0790-M [CD]), the PICS (joint grant between CNRS and the University of California at Davis), and an EEC contract between CNRS and Centro de Investigacion y de Estudios del I.P.N. (Mexico).
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