Stable RNA Modification
Chapter
57
GLENN R. BJÖRK
In Escherichia coli and in Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) there are several species of stable RNA of which the most prominent are ribosomal RNA (rRNA) and transfer RNA (tRNA). These two kinds of nucleic acids may, under certain physiological conditions, constitute more than 70% of the dry weight of these bacteria (195). Thus, a major part of the cellular biosynthetic capacity is devoted to the synthesis of stable RNAs. Most stable RNA, except 5S rRNA (41), contains modified nucleosides, which are derivatives of the four ordinary nucleosides. During maturation of both rRNA and tRNA, specific nucleosides are modified by enzymes that can be considered rRNA and tRNA biosynthetic enzymes, respectively. All modifications, except one (queuosine [Q]), are made following the synthesis of the primary transcript; i.e., modification occurs at the polynucleotide level. Figure 1 shows the structures of a few of the more than 80 modified nucleosides that have been characterized so far in tRNA and rRNA from various organisms (77, 167, 176).
Structures of different modified nucleosides as well as their abbreviations are described in reference 176. An index and an exponent indicate the number and the position of the substitution, respectively; e.g., 6-dimethyladenosine is abbreviated 
A. c-, i-, k-, m-, n-, o-, r-, s-, and t- are abbreviations of carbonyl, isopentenyl, lysine, methyl, amino, oxy, ribose, thio, and threonine groups, respectively. acp- denotes 3-amino-3-carboxypropyl. An abbreviation to the left or to the right of the nucleoside symbol denotes a modification of the base and the ribose, respectively. Other abbreviations: ψ, pseudouridine; I, inosine; Q, queuosine; oQ, epoxyqueuosine; R, purine; Y, pyrimidine; and N, any of the four major nucleosides. A number following an abbreviation for a modified nucleoside denotes the location of it in rRNA or tRNA. An enzyme catalyzing the formation of m5U at position 54 in the tRNA is denoted tRNA(m5U54)methyltransferase, and likewise for other tRNA- and rRNA-modifying enzymes. Methionyl-tRNA synthetase is denoted MetRS, an unspecified aminoacyl-tRNA synthetase is denoted AaRS, and the peptide encoded by the trmA gene is denoted TrmA and likewise for other aminoacyl-tRNA synthetases and other gene products. tRNA species are identified by their anticodon sequence.
In 16S rRNA there are 10 methylated nucleosides (52, 304; reviewed in reference 237) and one ψ (15, 75, 102, 170, 196; J. A. Kowalak and J. A. McCloskey, personal communication) (Fig. 2). The large majority of these modified nucleosides are base methylated, and all but one are found in single-stranded regions with a conserved primary structure. Five (m4Cm, m5C, m3U, m2G, and A) of the 10 methylated nucleosides in 16S rRNA are present in the 3'-proximal hairpin loop structure (Fig. 2), which is one of the most conserved regions of the rRNA. In 23S rRNA there are 23 modified nucleosides (16, 36, 205, 271), of which most also are located in conserved regions. Figure 2 shows that all 11 modified nucleosides in 16S rRNA are clustered in the ribosomal decoding site and 20 out of the 23 modified nucleosides identified so far in 23S rRNA are clustered in the peptidyltransferase center (39, 40). One (Gm2251) of the three 23S rRNA modifications located outside this region may be part of the peptidyltransferase center, since Gm2251 is found next to where P-site-bound tRNA protects rRNA from chemical modifications (188). Thus, 31 to 32 of the 34 modified nucleosides so far identified in rRNA are clustered in or close to the functional centers of the ribosome. This striking modification pattern in both 16S and 23S rRNA suggests a pivotal role of modified nucleosides in the translational activity.
The methylation of 16S rRNA during the assembly of 30S ribosomes in vivo is a late event (68, 75, 92, 137, 179, 273, 289), and the assembly process is independent of modification (20, 21, 56). Accordingly, unmodified in vitro synthesized 16S rRNA assembles into 30S subunits in vitro (170, 183). However, the conditions required for the assembly are more extreme than those for native 16S rRNA, the particles are more heterodisperse in size, and their biological activity is only half of that of controls (65). Still, the methylation must take place at different stages during the maturation of 16S rRNA, since some methyl groups (m7G, m4Cm) present in mature 16S rRNA are lacking in the immature 17S precursor rRNA isolated from the 27S immediate precursor particle of the 30S ribosomal subunit (137, 273). Furthermore, the formation of A at positions 1518 and 1519 must occur after the assembly of the 30S subunit, since the rRNA(A)methyltransferase requires a 30S subunit as substrate (227). In vitro, the formation of m5C967 is an early event as it can occur on naked 16S rRNA and is blocked by the addition of protein S19. The binding of S19, on the other hand, induces the formation of m2G at the nearby position (966), indicating a temporal synthesis of these two methylated nucleosides (194, 310). Thus, although the modification is not required, it facilitates the assembly of the 30S subunit, and a sequential modification occurs that is an integral part of this process. Therefore, it is reasonable to assume that if the assembly process is disturbed, e.g., by a mutation that alters a ribosomal protein, undermodification of the rRNA can occur (249).
The primary transcript from the rrn operons is a 30S rRNA precursor, which contains the sequences of 23S, 16S, and 5S rRNA (76, 104, 138, 200). Whereas the 16S rRNA counterpart in the 30S rRNA precursor is completely unmodified, the 23S rRNA counterpart is extensively modified (68). Thus, in contrast to the modification of 16S rRNA, the modification of 23S rRNA is an early event. Still, some methylation must occur late, since the conversion of the 43S precursor particle into a mature 50S subunit requires methylation (198). Three precursor particles (32S, 43S, and near 50S) are successively formed in vivo during the assembly of the 50S ribosomal subunit (see review in reference 197). Treatment by ethionine, which inhibits methylation, results in incompletely maturated 50S-like particles lacking L16 and containing reduced amounts of several other ribosomal proteins. Such particles are functionally inactive in protein synthesis and contain hypomethylated 23S rRNA (5). These results show that methylation of rRNA and/or ribosomal proteins is required for a proper assembly. Accordingly, 50S particles have so far not been assembled in vitro using unmodified 23S rRNA transcript. Therefore, at least one methylated nucleoside of the 23S rRNA may be required for the 50S assembly. Although the modification of 23S rRNA is an early event in vivo, some enzymes, like the 23S rRNA (m1G745)methyltransferase, can use both naked 23S rRNA and 70S or 50S ribosome subunits as substrates in vitro (147). Therefore, some modification sites are on the surface of the ribosome and are accessible for modification, suggesting that even after various proteins have bound some modifications are not blocked.
Six of the rRNA-modifying enzymes (formation of m1G, m2G, m6A in 23S rRNA; A1518; A1519, m2G966, and m5C967 in 16S rRNA) have been partially purified (148, 194, 227, 310). In addition, in experiments using 16S rRNA or 23S rRNA transcripts or mutant rRNA, enzymes catalyzing the formation of m3U and m6A in 16S rRNA and m5C, m2G, and m5U in 23S rRNA have been observed (29, 194, 311). Only the rRNA(A)methyltransferase, which methylates the two A’s at position 1518 and 1519 in 16S rRNA, has been purified to homogeneity (227). This enzyme can only use 30S subunit as substrate and is able to methylate either of the two A residues in the absence of the other, ruling out any obligate order of methylation of A1518 and A1519 (66). The 23S rRNA(m2G)methyltransferase uses an assembled 23S rRNA as substrate, whereas the 23S rRNA(m1G745)methyltransferase is able to use either 50S, 70S, or 23S rRNA as substrate (147, 148). Thus, most rRNA-modifying enzymes use more or less matured, high-molecular-weight rRNA or subunits as substrates. One enzyme, however, the 23S rRNA(m6A)methyltransferase, which catalyzes the formation of m6A in 23S rRNA, can also use as substrate the low-molecular-weight compound β-9-ribosyl-2,6-diaminopurine, which is an adenosine analog (6, 267).
Assuming that one enzyme is required for each of the modified nucleosides present in 16S and 23S rRNA, at least 36 rRNA-modifying enzymes would be present in E. coli and S. typhimurium. The genetic information needed for these enzymes is about 36 kb, which would represent about 0.8% of the genetic information in these bacteria, i.e., similar to that required for the seven rrn operons (about 32 kb). Mutants defective in the synthesis of m2G, m5C, and m1G (rrmA) in 23S rRNA have been characterized but not located on the chromosome (28, 29). The ksgA mutant, which is resistant to the antibiotic kasugamycin, lacks the A at positions 1518 and 1519 in 16S rRNA (140). The ksgA gene, the DNA sequence of which has been determined (303), is located at 0.9 min on the E. coli chromosome (7, 276). The rRNA(A)methyltransferase is autogenously regulated at the level of translation (306). Thus, very little information exists about the genetics of rRNA modification and how the various enzymes are regulated.
The location of most of the modified nucleosides in or close to functional regions in both 16S and 23S rRNA (Fig. 2), usually at highly conserved regions, strongly suggests that these modifications play an important role in the activity of the ribosome. Methylation, except the formation of m7G, results in hydrophobic patches, and many of them are located at the interface between the subunits(186). On the other hand, formation of ψ creates an additional imino group compared to unmodified U (cf. Fig. 1). This results in hydrophilic groups that may be directly involved in the fixation and orientation of the ligands involved in translation. That may explain why so many ψs are located in the peptidyl center. Although the reconstituted 30S subunit containing completely unmodified 16S rRNA is not blocked in P- or A-site binding and peptide synthesis in vitro, the activity is reduced, suggesting a functional role in translation of the modified nucleosides in 16S rRNA (65). Similar overall tests of 23S rRNA modification have not been performed, since 50S subunits containing unmodified 23S rRNA have so far not been assembled. These as well as other results mentioned above suggest a functional role of the modification in the assembly. This suggestion is supported by a recent observation that the methylation to form the highly conserved Gm2251 in the large subunit rRNA from yeast mitochondria is essential for the assembly process (268). Thus, both the conserved location and these experimental results suggest an important function of rRNA modification in assembly and in translation. The synthesis of modified nucleosides in rRNA may have evolved as an optimization or a requirement for the catalytic activity of the rRNA (173, 235). Alternatively, the modification may prevent an endogenously unwanted folding pathway or unwanted catalytic activity of the rRNA.
Mutants lacking A (ksgA) in 16S rRNA or m1G (rrmA) in 23S rRNA, or deficient in m2G or m5C (23S rRNA), are all viable. The methylation resulting in the two As increases the stacking of these two adenosines (217, 288, 305), which in turn decreases the stability of the stem and thereby changes the local conformation of the molecule (141, 142). Deficiency of these two methylated adenosines decreases the affinity of 30S subunit to associate to 50S subunit in vitro and imposes a requirement for more initiation factor 3 (IF3) for in vitro fMet-tRNA binding to 30S subunit in the absence of initiation factor 1 (IF1) (228). These observations, as well as the location of A residues close to the Shine-Dalgarno sequence, suggest that they are important for the initiation of translation. This may be reasonable, since kasugamycin, to which A-deficient strains are resistant, inhibits the binding of fMet-tRNA to wild-type but not to mutant ribosomes, inhibits the initiation process by destabilizing the 30S initiation complex, and prevents the formation of 70S initiation complex (226). Thus, the two conserved A residues participate in some way in the initiation of translation. However, these two methylated adenosines may also participate in the elongation cycle, since lack of A induces increased leakiness of certain nonsense and frameshift mutations (302). Although lack of A in E. coli 16S rRNA only slightly reduces the growth rate (146, 307), the disruption of the yeast ksgA gene homolog (DIM1) revealed that this yeast gene is essential (172). This implies that the A has an essential role in the function of the yeast ribosome or that the DIM1 peptide has an additional unknown essential function.
The E. coli mutant (rrmA10) lacking m1G in its 23S rRNA is viable, but the mutant is resistant to the antibiotic viomycin (C. Gustafsson and H. Noller, personal communication). This antibiotic is known to bind to a region 170 nucleotides from the m1G745 in 23S rRNA, although in space this modified nucleoside may be much closer to the binding site of the antibiotic. Lack of this methylated nucleoside may therefore induce a local structural change preventing the binding of the antibiotic.
In summary, the clustering of 31 to 32 of the 34 modified nucleosides in the functional centers of the ribosome is striking and strongly suggests an important role in the decoding process. The modification of 16S rRNA is an early event, whereas 23S rRNA modification occurs late in the assembly process. Accordingly, some rRNA-modifying enzymes use as substrate naked rRNA, whereas others require a more or less assembled particle. The modification of 16S rRNA is not required for, but facilitates the assembly of the 30S subunit, whereas some modification of 23S rRNA may be an essential component in the formation of the mature 50S subunit. Although E. coli mutants defective in rRNA modification so far characterized are all viable, analysis of one such mutant has revealed that 
in 16S rRNA participates in both the initiation and the elongation steps of translation.
All 79 tRNA genes present in E. coli have been sequenced and located on the chromosome (166). These genes encode 46 different tRNA species, of which 37 E. coli species and 3 S. typhimurium tRNA species (Pro1, Pro2, and Pro3) have been sequenced. Thus, 40 of the expected 46 tRNA species present in E. coli or S. typhimurium have been characterized. Thirty-one different modified nucleosides have been located in tRNAs from E. coli and S. typhimurium, and only two of them are uncharacterized (Fig. 3). Therefore, at present we have an almost complete picture of the tRNA modification pattern in these organisms. Figure 3 shows that mainly single-stranded regions in the tRNA are modified. All tRNA species contain m5U54 and ψ55, and positions 34 and 37 are modified in 18 (45%) and 31 (78%) of the 40 sequenced tRNA species, respectively.
Some modified nucleosides (ψ, s4U, D) are found in more than one position of the tRNA (Fig. 3). It is known that the formation of ψ in the anticodon region (positions 38, 39, and 40) is catalyzed by the HisT enzyme, whereas another enzyme is responsible for the formation of ψ55 (265). It is therefore believed that most if not all tRNA-modifying enzymes are position specific. In seven cases, a certain modified nucleoside is found in only one tRNA species (ψ13 in 
; D21 in 
; m6A37 in 
; m6t6A37 in 
; ac4C34 in elongator 
; k2C34 in 
; and I34 in 
). The synthesis of each of these modified nucleosides requires, most likely, a specific modification enzyme. (A few other nucleosides, like i6A37, mnm5U34, or mnm5Um, are present in only one tRNA species, but their synthesis may utilize enzyme systems involved in the synthesis of other nucleosides, like ms2io6A37 or mnm5s2U34.) The bacteria thus seem to have several tRNA-modifying enzymes involved only in the modification of one specific tRNA species.
Modifications at positions 34 and 37 are not only frequent, but there is also a great variety of modified nucleosides found in these two positions (Fig. 3). In no case is an unmodified A present in position 34, and only 
, which has an unusual structure in many respects (257), has an unmodified U34. A certain pattern can be seen in the modification at position 34; e.g., Q and mnm5s2U34 are found in tRNAs reading codons of the type NAY and (C/A/G)AR, respectively (Fig. 4). A specific pattern is also found for modification in position 37 (Fig. 4). Codons that start with U or A are usually read by tRNAs having an i6A or t6A derivative in position 37, whereas codons starting with C or G are read by tRNAs having m1G, m2A, m6A, or an unmodified A in position 37 (Fig. 4). Unmodified A is found in 10 tRNA species, but unmodified G37 is always converted to m1G, implying that an unmodified G is detrimental for the tRNA. This is also supported by the fact that m1G37 prevents tRNA frameshift (see below, function). Although there is a good correlation between a specific modification at either position 34 or 37 and a specific coding capacity of the tRNA, it does not follow that the function of a particular modification is essential for all the tRNA species it is part of. The presence of the modification is also determined by the specificity of the corresponding tRNA-modifying enzyme. For example, the presence of t6A derivatives in the subset of tRNAs that read codons starting with A may impose a function that is qualitatively or quantitatively different among the various tRNA species having the positive recognition determinants (e.g., U36-A37) for the tRNA(t6A37)synthetase. In some tRNA species this modification may be neutral and could be present simply because the tRNA fulfills the requirement to be threonylated. Thus, the function of a specific modification may not necessarily be the same in all the tRNA species in which it is present.
Thirteen modified nucleosides, including m1G and t6A derivatives, are present in the same subset of tRNAs in all organisms, suggesting that the occurrence of these modified nucleosides was an early evolutionary event and that they perhaps even were present in the tRNA of the progenitor (25). Therefore, the function of these kinds of modified nucleosides may be the same irrespective of the origin of the tRNA.
Depending on the growth conditions, tRNA from E. coli and S. typhimurium may also have selenium-containing modified nucleosides, of which mnm5Se2U is the most prominent and likely to be present in tRNA specific for lysine and glutamate in position 34 (318, 319, 321).
There are 31 different modified nucleosides in the 40 sequenced tRNAs from E. coli and S. typhimurium, but the number of tRNA-modifying enzymes required for their synthesis is considerably higher. Some modified nucleosides, like ψ, are present in several positions, and their synthesis is catalyzed by position-specific enzymes. Furthermore, some are complex, like mnm5s2U34 and ms2io6A37, and more than one enzyme is involved in their synthesis. From such considerations one can infer that about 45 different tRNA-modifying enzymes are required for tRNA modification. Assuming the average size of a structural gene to be about 1 kb, the synthesis of these enzymes requires as much as 1% of the bacterial genome. The 46 tRNA species present in E. coli are coded for by 79 tRNA genes (166), and they represent about 0.25% of the genome size of E. coli. Thus, in bacteria at least four times more genetic information is devoted to the synthesis of the tRNA-modifying enzymes than to the synthesis of their substrate, the tRNA.
Of these 45 putative tRNA modification genes, 19 are identified, and their map locations are summarized in Table 1. Of these, nine have been sequenced, and with two exceptions (trmA and perhaps trmC), they are all part of multicistronic operons with complex regulation and transcription patterns; this has recently been reviewed (27). An overview is shown in Fig. 5.
Table 1Mutations that affect tRNA modification |
The hisT gene is the structural gene for the tRNA(ψ38,39,40)synthetase (also called tRNA pseudouridine synthetase I) (55, 62, 265), which catalyzes the formation of ψ38, ψ39, and ψ40 in many different tRNA species in E. coli and S. typhimurium (299). The hisT operon may consist of up to six unrelated genes (10, 32, 206, 256) (Fig. 5). The hisT gene is translationally coupled with the upstream asd gene, which gene product is, however, made in 10 to 14 times higher amount than the HisT peptide (9). This peptide, as well as its mRNA, is growth rate-dependently regulated, and its transcription is induced when cells are entering the stationary growth phase (298).
The trmA gene encodes the tRNA(m5U54)methyltransferase, and its expression is growth rate and stringently regulated (121, 209, 210). Indeed, the lowly expressed trmA gene is regulated in a way similar to the highly expressed rrn genes (encoding rRNA) and to many of the tRNA genes. There is an extensive similarity between the trmA and the rrn P1 promoters (121) including the GC-rich region located between the –10 Pribnow box and the +1 transcriptional start site, which is typical of stringently controlled promoters (the "discriminator") (294). Furthermore, the sequence TCCC located just upstream of the –10 region is present in the trmA promoter, in all seven rrn P1 promoters, and in some tRNA promoters, but not in any other σ 70 promoter (121). Although the significance of this TCCC sequence in the trmA promoter is not clear, the TCCC motif in the rrnB P1 promoter is controlling both the level of expression and the growth rate-dependent regulation, perhaps in conjunction with the discriminator (73, 98). These features of the trmA promoter could explain, at least partly, the similarity in regulation of the rrn and trmA genes (121, 209, 210). Why the expression of the trmA and the rrn genes is regulated similarly is not clear, but it results in a constant ratio of the tRNA(m5U54)methyltransferase and the amount of bulk tRNA and rRNA. Although this regulation of trmA gene expression results in a balance of the level of tRNA(m5U54)methyltransferase and bulk tRNA, each tRNA species, all of which contain m5U54, is differentially regulated (83). One possible reason for the coordinated regulation of the synthesis of tRNA(m5U54)methyltransferase with that of rRNA and bulk tRNA could be that this enzyme has an unusual feature: it exists in two forms, the native TrmA peptide and a form in which the TrmA peptide is covalently bound to rRNA/tRNA (120).
Three genes, miaA, miaB, and miaE, whose gene products all are involved in the synthesis of ms2io6A37, are not present in the same operon but scattered on the bacterial chromosome (Table 1). The gene organization for two of them, the miaA and miaE genes, has been established. The miaA gene, which is the structural gene for the tRNA(i6A37)synthetase, is part of a complex operon (Fig. 5) consisting of probably six unrelated genes, of which the first two genes encode proteins with unknown functions; the third gene, amiB, may encode a periplasmic N-acetyl-muramoyl-l-alanine amidase (297); the fourth gene is mutL, which encodes a protein involved in mismatch repair; the fifth gene is miaA (49, 61); and the sixth gene is hfq, which encodes the host factor, HF-1, required for phage Qβ RNA-directed synthesis (155). Transcription of the miaA operon is governed by several internal promoters. One internal promoter exists within the mutL gene upstream of the miaA gene, and three are located within the miaA gene and govern the transcription of the last gene hfq (297). Furthermore, expression of the mutL and miaA genes is translationally coupled (61). The miaE gene, which is responsible for the hydroxylation of ms2i6A37 to ms2io6A37 in tRNA from S. typhimurium, is the second gene in a dicistronic operon. The transcription of the miaE gene is very low due to a strong (90 to 95%) transcriptional attenuator present between the first gene and the miaE gene. The miaE gene is absent in E. coli, consistent with the lack of the hydroxylated derivative of ms2i6A in its tRNA (222).
The trmD operon (Fig. 5) consists of four genes: rpsP, which encodes the ribosomal protein S16; 21K, which encodes a 21-kDa protein that influences the activity of the ribosome (B. C. Persson, Ph.D. thesis, Umeå University, Umeå, Sweden, 1993); trmD, which encodes the tRNA(m1G37)methyltransferase; and rplS, which encodes the ribosomal protein L19. These four genes are transcribed in the above order as a single polycistronic mRNA (47, 48) (Fig. 5). Just upstream of the promoter-proximal gene rpsP (S16), an attenuator-like structure exists, which in vitro causes 70% of the transcripts to terminate. There are no internal promoters or processing sites (48). Like other ribosomal protein operons (177), the accumulation of the mRNA from the trmD operon is growth rate-dependently and stringently controlled (48). However, unlike other ribosomal protein operons, the trmD operon is not subjected to translational and transcriptional feedback regulation (314). The rate of synthesis of the four proteins is significantly different, resulting in a 12- and 40-fold lower amount of the 21K protein and tRNA(m1G37)methyltransferase, respectively, as compared to the amount of the two ribosomal proteins, flanking the 21K and trmD genes (313). This difference in expression is achieved by regulation at the translational level. A large stem-and-loop structure is formed by mRNA sequences 100 nucleotides downstream from the start codon of the 21K gene that fold back and base pair to the translational start site of the 21K mRNA. The stem-and-loop structure prevents entry of the ribosome and thus decreases the frequency of translation initiation. A similar stem-and-loop structure, which inhibits the initiation of translation, is also present at the beginning of the trmD gene (315). The tRNA(m1G37)methyltransferase activity is invariant with growth rate, although the amount of mRNA transcript and the TrmD polypeptide increases with growth rate (48, 210, 212, 313). Neither the enzymatic activity (212) nor the synthesis of the polypeptide (313) is stringently regulated, in contrast to the accumulation of the mRNA (48). This discrepancy between transcriptional and translational regulation suggests that a regulatory device is operating at the translational level. Clearly, the trmD operon exhibits many complex regulatory features resulting in different and noncoordinate translational regulation of gene expression of the four genes of the operon.
Although three mutants defective in the biosynthesis of Q have been identified, only two genes, queA and tgt, have been localized on the chromosome and sequenced (203, 239). Both genes are part of the same operon, which consists of five cistrons. The first gene (queA) in this operon is preceded by the main promoter (PM), but two internal promoters (PI and PII) also exist. However, transcription initiated at PM is predominantly responsible for the expression of the queA and tgt gene products (270). The different transcripts initiated at the three promoters may all terminate after the secF gene, since the only rho-independent transcriptional terminator present in the operon is located downstream of the fifth gene, secF. Interestingly, the first promoter (PM) contains a "discriminator," which implies that the expression from this promoter, as for the trmA, trmD, and hisT promoters, is stringently regulated. A high-affinity binding site for the FIS (factor inversion stimulation) protein is centered around nucleotide –58, a location similar to the putative FIS binding site in the trmA promoter. The FIS binding site is located in an upstream activating sequence, which increases the transcription of the queA gene twofold (270).
tRNA-Modifying Enzymes.
s4U8,9. The thiolation of U8, present in many unmaturated tRNAs, and presumably also U9, present only in unmature tRNATyr, occurs by two enzymatic reactions (1). Accordingly, two genes, nuvA and nuvC, have been identified as responsible for the synthesis of s4U in tRNA (178). The NuvA protein converts the tRNA in an ATP- and Mg2+-dependent manner into an unidentified intermediate, perhaps an activated 4-hydroxyl group of U8(U9). The enzyme is only involved in the synthesis of s4U and not in the synthesis of any other thiolated nucleosides (292). The NuvC protein, a cysteine-s4U8 sulfur transferase, catalyzes the second step, which is a transfer of a sulfide from cysteine to the unidentified intermediate, resulting in the formation of s4U. In addition to the lack of s4U8, a mutation in the nuvC gene also results in auxotrophy for thiamine (248). Therefore, the NuvC protein may also transfer sulfur to an intermediate in the thiazole biosynthesis. Although the NuvC peptide also participates in the synthesis of thiamine, the synthesis of no other thiolated nucleoside is affected by a mutation in the nuvC gene (248). The key event may be that the NuvC peptide generates a suitable activated low-molecular-weight sulfur donor. If so, the NuvC peptide is in some aspects analogous to the SelD peptide, which also generates an activated donor molecule that participates in two different pathways, the selenation of mnm5s2U to mnm5Se2U and selenation of Ser-
to Sec- (see below). Thus, thiolation and selenation of a uridine in tRNA may proceed with similar mechanisms.
mnm5s2U34. tRNAs which read codons of the split codon boxes (a codon box in which the four codons encode two amino acids) have in position 34 (wobble position) modified uridines of the type xm5U or xm5s2U (x can be amino, methylamino, or carboxymethylamino groups). The thiolated derivatives mnm5s2U or cmnm5s2U (S. typhimurium) are present in tRNAs specific for Gln, Lys, and Glu, whereas the nonthiolated derivative cmnm5U34 is present in one of the tRNALeu species (282a) and mnm5U34 in one of the tRNAArg species (250, 275) (Fig. 3 and 4). Although these two modified nucleosides do not have an s2 group, they may still be synthesized by the same enzymes as those involved in the synthesis of the thiolated derivatives. The synthesis of mnm5s2U34 may occur in the following steps:
An asuE mutant contains mnm5U (281; T. Hagervall and J. McCloskey, personal communication). Thus, the modification at position 5 of the base occurs independently of thiolation (281), and the thiolation reaction is therefore not necessarily the first reaction as depicted above. Only one of the two-carbon atoms present in mnm5s2U34 originates from S-adenosylmethionine (AdoMet), and the first step in the synthesis of the mnm5 side chain is not an AdoMet-dependent methylation (82, 123). The methylene group may therefore come from tetrahydrofolate (287). The homogeneously purified tRNA(mnm5s2U34)methyltransferase (123, 286, 287), which is a 79-kDa protein, first converts cmnm5s2U34 to nm5s2U34 and then methylates nm5s2U34 to mnm5s2U34 in the presence of AdoMet (123). Therefore, the TrmC peptide has two enzymatic activities, and the two trmC1 and trmC2 mutations affect these two activities, respectively.
mnm5Se2U34. When selenium is available, 40% of mnm5s2U34 is replaced by the selenium derivative, mnm5Se2U34, in E. coli and S. typhimurium tRNAs specific for Glu, Gln, and Lys (168, 320). The incorporation of Se into the wobble base of these tRNAs occurs by an ATP-dependent process that involves the replacement of the sulfur of mnm5s2U with Se (320). A mutation in the selD (designated selA in S. typhimurium) gene prevents incorporation of selenium not only into tRNAs but also into selenocysteine-containing proteins (168, 277). Therefore, the SelD protein serves as a general selenium transfer enzyme. A pure SelD protein catalyzes a selenium-dependent ATP cleavage that results in AMP, Pi, and the labile selenium donor molecule phosphoselenoate (H2PO3SeH (78, 308). This selenium compound is then used by a tRNA selenation enzyme to replace the sulfur atom of an s2U derivative with selenium. If so, the synthesis of selenium nucleoside is reminiscent of the thiolation of U8 forming s4U8, which also occurs in two steps.
Q34. The hypermodified nucleoside Q (the base is designated queuine) consists of a 7-(aminomethyl)-7-deazaguanine, called the base of preQ1, to which a cyclopentenediol is linked to the NH2 group (Fig. 1). The enzyme tRNA (preQ134)transglycosylase from E. coli is a 45-kDa polypeptide encoded by the tgt gene (203, 214) and has a somewhat broad substrate specificity, since it in vitro efficiently inserts Gua, the 7-(aminomethyl)-7-deazaguanine (base of preQ1), or the 7-cyano-7-deazaguanine (base of preQ0), but not queuine (base of Q), into the polynucleotide chain. This insertion occurs by cleavage of the N-C glycosyl bond without breakage of the phosphodiester bond (215). The synthesis of Q in E. coli tRNA may be as shown in Fig. 6.
A queA mutant accumulates preQ1 in its tRNA (216, 239), whereas another mutant (here denoted queB) accumulates a precursor, 7-(cyano)-deazaguanosine (preQ0), in its tRNA (204). The base of preQ1, and not the base of preQ0, is likely the normal substrate in vivo for the Tgt enzyme, since it is found in free form in E. coli and accumulates as such also in a tgt mutant (203, 215). If so, the base of preQ0 may be a precursor to the base of preQ1 synthesized prior to the insertion of the base of preQ1 into the tRNA (204). Since queuine (base of Q) is not a substrate for the tRNA(preQ1)transglycosylase, the cyclopentenediol is added after the base of preQ1 is incorporated in tRNA. Accordingly, there is an enzyme, the QueA protein, that transfers and isomerizes the ribose moiety of AdoMet to the epoxycyclopentane of oQ (269). A later step in the synthesis of Q is the reduction of the intermediate oQ to Q, a step that requires vitamin B12 (97).
Only tRNAs specific for Tyr, His, Asn, and Asp have Q34. This subset of tRNAs recognizes U or C in the third position of the codon and A in the second (Fig. 4). Therefore, the tRNA(preQ1)transglycosylase has a strong requirement for the nucleotides on both sides of the target nucleotide (53), although other structural features, like the bases present in positions 36, 37, and 38, also contribute to the activity (136). The strict requirement for U35 is consistent with the presence of Q in the aforementioned subset of tRNAs. Indeed, a 17-base oligonucleotide corresponding to the anticodon loop and stem serves as a fairly good substrate for the tRNA transglycosylase from E. coli, indicating that the positive recognition elements for this enzyme are located in the anticodon loop and stem region (67). Thus, the synthesis of Q and its derivatives shows a strong requirement for a specific sequence around the target nucleoside.
cmo5U/mcmo5U34. Synthesis of the modified nucleosides cmo5U and mcmo5U34 requires chorismic acid, and only one of the two carbon atoms originates from AdoMet (24, 126). The tRNA methyltransferase, which catalyzes the last step in the synthesis of mcmo5U34, has been purified (230). The enzymatic activity is absent in UGA suppressor (supK) strains (238), suggesting that the supK gene is the structural gene for the enzyme. However, the supK gene is the structural gene for release factor 2. How a mutation in release factor 2 could affect the amount of the tRNA(mcmo5U34)methyltransferase or its activity is not understood (157).
m1G37. The tRNA(m1G37)methyltransferase from E. coli is a 28-kDa protein (47, 143) and is present in only 260 molecules per genome at a growth rate of k = 1.0 (313). Seven of the 46 different tRNA species present in E. coli are methylated by this enzyme. These seven tRNAs read leucine (CUN), proline (CCN), and arginine (CGG) codons, and consequently, the anticodons of these tRNAs all have G36. In the case of E. coli 
(anticodon C34-A35-G36), elimination of any of the three loops (V-, D-, or T-loops) reduces the V max 10-fold, but the enzyme efficiently binds such defective substrates in all cases except when the T-loop is eliminated (144). The enzyme also senses the stacking interaction in the anticodon stem. Changes in position 38, i.e., 3' of the target nucleoside, have little effect on the kinetic parameters. This is in sharp contrast to the effect of base substitutions in position 36, which result in a 20-fold decrease in the V max. Thus, the main determinants of the tRNA(m1G37)methyltransferase for the recognition of 
are G36, a strong stacking interaction in the anticodon stem, and the overall structure of the tRNA. Interestingly, most changes that decrease the V max also decrease the Km, suggesting that the enzyme still binds the altered substrates but cannot efficiently catalyze the transfer of the methyl group. Analyses of mutant variants of bacterial 
isolated in vivo also suggest that the overall structure of the tRNA is important for substrate recognition by the bacterial tRNA(m1G37)methyltransferase (Q. Qian and G. R. Björk, unpublished observations).
i6A37, ms2i6A37, and ms2io6A37. The hydrophobic nucleosides i6A37, ms2i6A37, and ms2io6A37 are present in tRNA reading codons starting with U (Fig. 3). The synthesis may occur in the following steps:
(The miaD gene was thought to regulate the synthesis of ms2i6A37 in E. coli [61], but was later shown to be allelic to the prfC gene, which is the structural gene for release factor 3 [107, 185].)
The isopentenyl group (i6) is derived from mevalonic acid (94, 224; see reviews by Hall [129, 130]), which is the precursor to many different metabolic products through its conversion to isopentenylpyrophosphate. The E. coli tRNA(i6A37)synthetase is encoded by the miaA gene (49, 60) and has been partially purified (18, 243). The enzyme transfers an isopentenyl group from isopentenylpyrophosphate to A37 in tRNA (93). The sulfur group originates from cysteine, and the methyl group originates from AdoMet (100).
The postulated biosynthetic pathway is derived from precursor analyses of methionine- or cysteine-starved E. coli cells (2) and analyses of mutants defective in the pathway (79, 86, 222). A mutation in the miaA gene results in the accumulation of unmodified A37 in the tRNA, but no ms2A37 is present. Thus, the MiaB enzyme has a strict requirement for the isopentenyl group, which is also consistent with some early experiments on the biosynthesis of ms2i6A (reviewed in references 129 and 130). However, the MiaB enzyme must require additional structural features, since 
, which has several unusual features, lacks the ms2 group but has i6A37 (257). The starvation of E. coli (rel met cys) for methionine (2) results in accumulation of a precursor to ms2i6A, which may be s2i6A37, whereas starvation for cysteine (2) or iron (108, 244, 312) results in accumulation of i6A. The last step requires the miaE gene, which is absent in E. coli (222). The hydroxylation reaction in S. typhimurium has a strong requirement for the ms2 group, since cysteine or iron starvation as well as a mutation in the miaB gene results in the accumulation of i6A37 and only a small portion (5 to 16%) is in the hydroxylated form (io6A37) (43; B. Esberg and G. R. Björk, unpublished observations). Therefore, the modifying enzymes synthesizing ms2io6A act strictly sequentially, and they are dependent on modifications at other positions of the nucleoside.
Comparison of sequences of tRNAs containing i6A derivatives suggests that the recognition determinants for the tRNA(i6A37)synthetase are the sequence A36-A37-A38 and a 5-bp anticodon stem (296). Analyses of various missense (51) and nonsense (234) suppressor mutant tRNAs support the suggestion that U36 is an important determinant for the isopentenylation reaction. Seryl-tRNA species I and V from E. coli have the sequence A36-A37-A38 but do not have ms2i6A37 (113). However, these tRNAs do not contain the 5-bp anticodon stem, which is the other postulated identity element. An insertion of an extra A in the anticodon loop of tRNAGly results in a primary sequence as present in tRNATrp, which has ms2i6A37. However, this extra base, in the mutant tRNAGly, changes the conformation such that the tRNA(i6A37)synthetase is unable to recognize such tRNA. This is probably due to the extended anticodon loop, although the primary sequence of the recognition signal for the enzyme is present (233). Thus, the tRNA(i6A37)synthetase seems to have a strict sequence requirement for its substrate recognition, as well as for a correct anticodon conformation.
t6A derivatives. Threonine is used to synthesize t6A37 in vivo (57, 231). Bicarbonate and threonine are incorporated in vitro into tRNA in an ATP-dependent reaction (81, 171). Interestingly, the same enzyme may also incorporate glycine instead of threonine. The t6A derivatives are present in tRNAs reading codons starting with A, and sequence comparisons suggest that the sequence U36-A37-A38 is one of the determinants for the tRNA(t6A37)synthetase (296). Analyses of various missense (241) and nonsense (232) tRNA suppressors, which have alterations in the U36-A37-A38 sequence, have demonstrated that U in position 36 is important for the formation of t6A, consistent with the requirements for the tRNA(t6A37)synthetase from other organisms (110, 111). The initiator 
present in E. coli contains the sequence U36-A37-A38 but does not contain t6A (278), which may suggest that the unusual anticodon structure of the initiator tRNA (322) is unfavorable to the tRNA(t6A37)synthetase. Thus, this enzyme has a strong requirement for U36, but other structural features of the anticodon are of importance.
ψ38, 39, and 40. The tRNA(ψ38,39,40)synthetase has been purified to homogeneity from E. coli (156) and partially from S. typhimurium (8). The enzyme binds strongly, but reversibly, to substrate tRNAs as well as to nonsubstrate tRNA species. The first step in the catalysis may be a nonspecific binding of tRNA, which is followed by a cleavage of the glycosyl bond, a rotation of the base relative to the sugar so that the C5 of the uracil is juxtaposed with the C1' of the ribose (see Fig. 1), and then formation of the C1' to C5 carbon-carbon bond of ψ. Ivanetich and Santi (150) have proposed a mechanism reminiscent of the one used by the tRNA(m5U54)methyltransferase (see below). They postulate a covalent intermediate in which the tRNA(ψ38,39,40)synthetase binds through one of the cysteines in the enzyme to the 6-carbon of uracil in the tRNA during the catalysis.
m7G46. The tRNA(m7G46)methyltransferase, which may be encoded by the trmB gene (181), has been partially purified (12). Following a 1,000-fold enrichment, the enzyme is present in two forms, 100 and 300 kDa, as revealed by gel filtration.
m5U54. The tRNA(m5U54)methyltransferase from E. coli is a 42-kDa polypeptide, which is consistent with the nucleotide sequence of its structural gene, trmA (106, 118, 121, 211). However, Ny et al. (211) also recovered additional forms of the enzyme that were associated with RNA. The RNA binds covalently to the enzyme and consists of a piece of the 3' end of 16S rRNA and a subset of undermodified tRNAs (120). As much as 50% of the tRNA(m5U54)methyltransferase molecules present in the bacterium are bound covalently to rRNA and/or tRNA. In logarithmically growing cells the enzyme is present in three forms: a 42-kDa native form, a 54-kDa TrmA-RNA complex, and a 62-kDa TrmA-RNA complex. Only the 54-kDa TrmA-RNA complex is accessible for phosphorylation by T4 polynucleotide kinase (120). Although the reason for the presence of these RNA-TrmA complexes is not understood, it may be related to an unknown second function of the TrmA peptide that is essential for cell viability (223).
The reaction catalyzed by tRNA(m5U54)methyltransferase proceeds by three distinct steps including the formation of a covalent bond between Cys-324 in the TrmA peptide and C-6 of U54 of the tRNA (see Fig. 7 for details). The U54 is buried in tRNA through stacking between G53 and ψ55. Since tRNA lacking only m5U54 but otherwise fully modified is a good substrate in vitro (28), the tRNA(m5U54)methyltransferase must open up the T-loop to get access to U54 (159). Whether or not this is also true in vivo is not known, since it is conceivable that the addition of the methyl group to form m5U54 occurs before the T-loop is fully arranged in the way it is in the mature tRNA.
A 17-bp oligonucleotide representing positions 49 to 65 (i.e., the entire T-arm) accepts methyl groups in vitro with a k cat of 36% and a specificity constant (k cat/Km) of 6% of that of the complete tRNA (119). In fact, an 11-mer, which only has 2 bp in the stem, is also a substrate for the enzyme albeit at reduced efficiency as compared to the 17-mer. However, the 11-mer has the same Km as the 17-mer. From these results, together with a comparison of over 40 different E. coli tRNA species, a consensus sequence for the methylation includes only 11 nucleosides of the T-arm: the Pu52-Py62 and G53-C61 base pairs of the stem and next to the loop; the conserved nucleosides U54, U55, C56, and A58 of the loop; and the semiconserved bases Pu57 and Py60 also in the loop.
In summary. Recently, there has been much progress in our understanding of the synthesis of modified nucleosides. Some complex biosynthetic pathways (synthesis of s4U8,9, of xm5s2U34 and its selenium derivatives, of ms2io6A37, and of Q34) have been characterized to some degree. A detailed enzymatic mechanism (m5U54) has emerged, and information about the recognition determinants for some tRNA-modifying enzymes has been revealed. Enzymes synthesizing some modified nucleosides (e.g., Q34, m5U54, ms2io6A37, t6A37) seem to have a strict requirement for a sequence near the target nucleoside, whereas others (e.g., m1G37) do not. Those that have no sequence determinants preferentially recognize structures beyond the target nucleoside.
Metabolic-Induced Changes in tRNA Modification.
Deficiency in any of the precursors, methionine, cysteine, or threonine, results in tRNAs lacking methylated, thiolated, or threonylated nucleosides, respectively. Metabolic limitations such as starvation for leucine, histidine, arginine, or phosphate, or the addition of the protein inhibitors puromycin or chloramphenicol, results in the accumulation of uniquely undermodified tRNAs (95, 163, 164, 165). Unbalanced metabolism, as such, is probably not the primary reason for the appearance of undermodified tRNA, since amino acid limitation during balanced growth also induces undermodification of tRNA (290). Whereas the level of mnm5s2U34 in E. coli is invariant of growth rate, the level of s4U8 in some but not all tRNAs decreases with increasing growth rate (84). Furthermore, the formation of Q34 and ms2i6A37 is growth phase dependent (17, 266). Thus, at least a few tRNA modifications seem to be dependent on the growth rate, the growth phase of the bacteria, or other physiological stress conditions. A dominant mutation in the ilvU gene changes not only the regulatory behavior of the IleRS and resistance to the amino acid analog thiaisoleucine, but it also changes the relative abundance of tRNAIle and tRNAVal isoacceptors (91). The ilvU product may inhibit the modification of tRNAIle and tRNAVal, resulting in a changed distribution of isoaccepting tRNAs and derepression of IleRS. Thus, changes in the intermediary metabolism caused by the growth environment or genetically sensed stimulus may impose severe alterations in tRNA modification.
Three aromatic amino acids (Tyr, Trp, and Phe) and four vitamins (ubiquinone, folate, menaquinone, and enterochelin) all originate from the same precursor, chorismic acid. The four vitamins are pivotal in aerobic (ubiquinone) and anaerobic (menaquinone) electron transport, iron uptake (enterochelin), and C1 metabolism (folate). The synthesis of the ms2 group of ms2io6A37 (108, 244, 312) and the conversion of GTP to the base of preQ1 require iron (162). Thus, the level of enterochelin and consequently iron transport determines the level of these two modified nucleosides in tRNA. Interestingly, the deficiency of the ms2 group of the ms2io6A37 modification stimulates the transport of aromatic amino acids and increases, under certain stress conditions, the frequency of specific mutations (GC to TA transversions), as does a mutation in miaA (44, 60, 61). Therefore, undermodification of ms2io6A37 and Q34 may be an important adaptive mechanism for bacteria growing in iron-limited conditions. Indeed, it has been suggested that such an adaptation is important for the pathogenicity of the bacterium (109). Unexpectedly, the first step in the synthesis of cmo5U34/mcmo5U34 requires chorismic acid or a metabolite in a hitherto unknown biosynthetic branch from it (24, 126). Thus, the level of chorismic acid is critical for the synthesis of several modified nucleosides (ms2io6A37, mnm5s2U34, cmo5U, mcmo5U, Q). The degree of tRNA modification set by the level of chorismic acid may therefore regulate several parts of intermediary metabolism in a manner similar to how ms2io6A37 deficiency affects the transport of aromatic amino acids and the frequency of mutation.
Unlike most modified nucleosides, the thiolated derivatives, like s4U, have a spectrum that extends into the near-UV light (300 to 400 nm; λ max of s4U is 334 nm). These wavelengths are the part of sunlight known to induce growth delay and killing of bacteria. When bacteria are illuminated by near-UV light, the cessation of growth occurs well before cell death (151). However, this growth delay does not occur in bacteria (e.g., nuvA mutants) lacking s4U in their tRNA (169, 236, 292, 293). Such mutants are more easily killed by broad-band near-UV light than is the wild type, implying that s4U8 in tRNA protects the cell from such stress (169; see, however, reference 295). Upon irradiation either in vivo or in vitro, s4U8 is cross-linked to the nearby nucleoside C13 (90, 291), and such cross-linked tRNA triggers the accumulation of ppGpp (236). Since this nucleotide inhibits cell growth and rRNA synthesis (54), the growth delay is at least partly due to ppGpp accumulation. Moreover, s4U8-C13 cross-linking mediates the induction of some oxidative stress proteins, ppGpp-inducible proteins, and the dinucleotide ApppGpp (169). Accordingly, the nuvA mutant, which lacks s4U8 in its tRNA, is defective in the accumulation of these stress-induced metabolites, including the dinucleotide ApppGpp (169). Thus, s4U8 may act as a sensor of near-UV irradiation and may mediate, through the synthesis of ppGpp and ApppGpp, the induction of specific proteins as a cellular response to this stress (169). Prior exposure of cells to near-UV (300 to 400 nm) light antagonizes the mutagenic effect of UV (254 nm) light and inhibits subsequent induction of the SOS response by irradiation with UV light (review in reference 89). The SOS response is known to inhibit cell division, and it induces several DNA repair systems. This photoprotection by near-UV light requires the presence of s4U in tRNA (50, 89). Although s4U8 is the most prevalent thiolated nucleoside in tRNA, several others also exist, e.g., mnm5s2U34. Those thiolated nucleosides can also be part of the sensing mechanism for near-UV light, since ppGpp is also accumulated in a nuv mutant although not to the same extent as in a nuv + strain (293). Taken together, the results strongly support the hypothesis that s4U8 in tRNA and perhaps some other modified nucleosides act as sensors for near-UV light and mediate the cellular responses to protect the cell from such a stress.
Growth under oxygen limitation affects the synthesis of at least two modified nucleosides, Q and ms2io6A. Some bacteria, like S. typhimurium, synthesize vitamin B12 only under anaerobic conditions (152). The conversion of oQ (epoxy-Q) to Q requires vitamin B12 (97). Consequently, tRNA from bacteria grown aerobically in a medium lacking vitamin B12 has oQ34 in place of Q34. The hydroxylation of ms2i6A37 requires molecular oxygen, and accordingly ms2io6A37 occurs only in aerobically grown cultures of S. typhimurium (43). Thus, when bacteria are shifted between growth conditions which differ in the presence or absence of oxygen, changes in the modification of tRNA occur. Such changes may be involved in sensing the aerobiosis in S. typhimurium (43). Recently, a mutant (miaE) of S. typhimurium defective in the hydroxylation reaction has been isolated. However, no difference in growth response was observed between the mutant and the wild type upon shift from aerobic to anaerobic conditions, implying that this particular modification is not critical for such a shift. Unexpectedly, such a mutant is unable to grow on the citric acid cycle intermediates malate, fumarate, and succinate (222), implying a link between tRNA modification and the regulation of the central metabolism.
A mutation in hisT of E. coli or S. typhimurium has pleiotropic effects of which many, but not all, can be explained by its influence on tRNA-mediated attenuation (35, 37, 63, 240, 299). One such pleiotropic effect not easily reconciled with a tRNA-mediated attenuation mechanism is how ψ deficiency influences the synthesis of ppGpp. Starvation of a stringent (relA +) hisT mutant for histidine does not provoke ppGpp synthesis (274). In contrast, in the same hisT mutant, synthesis of ppGpp occurs upon starvation for serine, threonine, or arginine, whose cognate tRNAs do not contain ψ38, 39, or 40. This anomaly may be due to the fact that the ψ-deficient uncharged tRNAHis, which normally contains ψ38 and ψ39, either cannot bind to the ribosomal A-site (a prerequisite for the relA +-mediated ppGpp synthesis) or, if bound, is unable to activate the RelA protein to synthesize ppGpp. Nevertheless, such histidine starvation of a hisT mutant leads to an abrupt cessation of stable RNA synthesis in a relA + strain. Thus, histidine starvation of a hisT mutant provokes stringent response without ppGpp accumulation (274). However, starvation of a relA + hisT mutant for lysine, for which the cognate tRNA contains ψ39, results in accumulation of ppGpp (193). Apparently, ψ38, which is normally present in tRNAHis, is the key position where the presence and absence of a ψ determines whether synthesis of ppGpp occurs. Interestingly, the presence of ψ38 influences the activity of suppressor tRNAs (26) and the degree of misreading (219) more than the presence of ψ39. Therefore, these observations suggest that ψ located in the anticodon loop (position 38) influences the activity of the tRNA in the decoding process more than ψ located in the anticodon stem (positions 39 and 40). Mutations in hisT also affect the production of antibiotic peptides like microcins and colicin V in a manner that may indirectly relate to their effect on translation (242). Moreover, a mutation in the hisT gene reduces the capacity to derepress the ilv and leu operons by a mechanism which either bypasses or alters the attenuation control (38).
In summary, the level of tRNA modification is sensitive not only to genetic defects but also to metabolic stress conditions and developmental stages. There are several known examples in which read-through of nonsense codons or frameshifting acts as a regulatory device (14, 85, 134, 135, 301). The degree of modification can strongly affect such regulatory devices, since modified nucleosides in tRNA affect the efficiency of the tRNA, sense the codon context, and control the reading-frame maintenance (see below). Therefore, tRNA modification may be a sensor for various environmental or developmental signals, which directs the cellular response to such changes. If so, tRNA modification may act as a global regulatory device and link translation to the central and intermediary metabolism of the cell.
Using runoff in vitro transcription of a tRNA gene under the control of a promoter recognized by the phage T7 RNA polymerase, completely unmodified tRNA can be synthesized in quantities suitable for various physical and biological studies (251). This technique allows a direct comparison between native and completely unmodified tRNA in various tRNA interactions. Such comparisons of the kinetics of the aminoacylation reactions with cognate and noncognate AaRS revealed that among 14 different unmodified E. coli tRNAs, all except three (E. coli 
[S. Yokoyama, personal communication], 
[282], and 
[285]) accept the cognate amino acid. Thus, for most aminoacylation reactions the modified nucleosides are not a prerequisite in vitro. However, in all cases but one (E. coli 
), the cognate interactions with the unmodified species have kinetic characteristics that are different, albeit only slightly, from that of the fully modified species (data compiled in Table 1 of reference 26). This suggests that the modified nucleosides moderately affect, either directly or indirectly (through conformational changes), the recognition of the tRNA by the cognate AaRS. Under certain conditions, the unmodified yeast tRNAPhe shows a considerable changed kinetics of aminoacylation (251) and the unmodified tRNAPhe does not adopt the native conformation (58, 128). Also, unmodified yeast 
has an altered conformation and the D- and T-loop interaction is disrupted (221). Although the kinetic parameters of the unmodified are similar to those of the modified in the cognate interaction with AspRS, such unmodified is mischarged by ArgRS with considerable efficiency (220). The major effect is in the rate of mischarging (k cat), whereas the Km for unmodified in the noncognate interaction increases only twofold. Perret et al. (220) suggested that in the case of yeast , the modified nucleosides (most likely the m1G in position 37 [A. Garcia, Ph.D. thesis, Université Louis Pasteur, Strasbourg, France, 1990]) act as antideterminants. These results show that modifications change the structure of the tRNA in a way that influences the efficiency of the aminoacylation reaction and counteracts mischarging. However, the unmodified E. coli tRNAs, specific for methionine or valine, and the undermodified yeast 
are not mischarged (251, 259). Therefore, it is not a general role of modified nucleosides in tRNAs to act as an antideterminant.
Modified nucleosides in several positions (positions 8, 26, 32, 38, 39, 40, 46, 47, 54, and 55) play no significant role in the aminoacylation reaction (26). However, analyses using specific chemical labels of tRNAs or mutants lacking a modified nucleoside in tRNA have revealed that some modified nucleosides in certain positions have a direct role in the recognition process of AaRS.
Position 34 (the Wobble Position). Two isoleucine isoacceptors, 
and 
(minor), are present in E. coli. These tRNAs contain G34 as the wobble nucleoside and the modified nucleoside lysidine (k2C34 or L34), respectively. Synthesis of lysidine, a derivative of cytidine, occurs by addition of the amino acid lysine (K) at position 2 of the base cytosine (191). Even though the chemical structures of G34 (present in 
) and k2C34 (present in 
) are quite different, IleRS recognizes both species. When k2C34 is replaced in vitro with C34, the anticodon of (minor) is converted to CAU, which is the anticodon of 
. Such mutated 
(minor) is efficiently misacylated with Met (190), since the C34 or ac4C34 is a positive identity determinant for the MetRS (261, 279). Taken together, these results show that the modification of C34 to k2C34 in acts as a positive identity element for IleRS and as an antideterminant towards MetRS to prevent a detrimental misacylation. In addition, variants of , which contain A34, U34, or C34 in place of G34, are not charged with isoleucine (189). Thus, the IleRS from E. coli must recognize as identity elements structural features that are common to the chemically different nucleosides G34 and k2C34.
Chemical modification by cyanobromide of mnm5s2U34 present in tRNAGln, tRNAGlu, and tRNALys significantly reduces the amino acid acceptance activity of these tRNAs (4, 160, 254, 264, 316). An unmodified tRNALys transcript, which lacks modified nucleosides (among them mnm5s2U34), has a 140-fold lower lysine acceptance activity compared to native tRNALys (285). Furthermore, absence of mnm5s2U34 in tRNAGlu reduces the specificity constant 15-fold (282). Moreover, the GluRS forms an H-bond with mnm5s2U34 (316). These results imply a direct interaction between the mnm5 and the s2 groups of mnm5s2U34 and the corresponding AaRS, which is consistent with the X-ray analysis of the GlnRS-
complex (246). Detailed analysis of the interaction between the enzyme and the anticodon revealed that the 2-thio group of mnm5s2U34 binds in the same way as the 2-keto group of C34, which is present in one of the two glutamine isoacceptors (247). This may explain why different chemical, physiological, and genetic treatments, which replace the s2 group of mnm5s2U34 by oxygen, do not affect the aminoacylation reaction (4, 263, 264, 282). Thus, the mnm5 group but not the s2 group is a positive determinant for LysRS, GluRS, and GlnRS from E. coli. Since the trmE mutant, for which the tRNA contains s2U34 instead of mnm5s2U34 (82), grows, albeit at a reduced rate, lack of the mnm5 group cannot completely abolish the aminoacylation reaction in vivo.
Table 2Function of modified nucleosides in tRNAa |
Position 37 (3' of the Anticodon).
Various chemical treatments of ms2i6A37 in E. coli tRNAPhe, tRNATyr, or tRNASer do not influence the aminoacylation kinetics (88, 105, 139). tRNAs specific for Tyr or Phe, containing unmodified A37, i6A37, or fully modified ms2i6A37, show the same aminoacylation kinetics (45, 101, 317). Thus, the ms2i6A37 is not important for the cognate aminoacylation reaction.
Completely unmodified 
has a much lower amino acid accepting activity than the native (189), suggesting that tRNA modification plays a crucial role in the recognition by IleRS. Specific replacement in vitro of t6A37 by A37 in the native drastically reduces the Ile accepting activity (199). Thus, t6A37 is a strong positive determinant for IleRS. However, unmodified 
, which normally also contains a t6A derivative, m6t6A37, has kinetic characteristics similar to those of native 
(133, 260). Thus, t6A37 is not an identity element for ThrRS. Apparently, the IleRS and ThrRS have different identity elements, which is also true for other aminoacyl-tRNA synthetases(103, 208, 258)
In Summary.
Nucleosides in the anticodon are important identity determinants for many aminoacyl-tRNA synthetases (103, 208, 258). Therefore, the involvement of k2C34 in the identity of 
and of mnm5s2U34 in the identity of 
, , and 
is not surprising. Still, some modified nucleosides in position 34, like ac4C34 in 
(279), I34 (285) and perhaps mnm5U in 
(285), and Q34 (203), have no or only minor effect on the aminoacylation reaction. Modification in position 37 may be (t6A37) or is not [ms2i(o)6A37] involved as determinant or antideterminant in the recognition process. The lack of influence of other positions (e.g., in position 54 in the case of m5U54) is reasonable, since these positions are not directly involved in the recognition process. However, these studies also show that when many modified nucleosides are absent, conformational changes occur that influence the kinetic parameters, and this may strongly influence the misacylation activities. This suggests that the conformational changes caused by the presence of modified nucleosides are indirectly involved as identity determinants. In one case (k2C34 in ), the modified nucleoside acts as an antideterminant.
It is apparent that certain positions in tRNA are more prone to be modified than others (Fig. 3). Especially, the anticodon loop is not only frequently modified, but there are also many different modified nucleosides found in this region of the tRNA, suggesting a direct and specific role of these modified nucleosides in the anticodon-codon interaction. The coding information of the tRNA may in fact extend beyond the anticodon towards the 3' side of the tRNA as far as to position 44 (325).
The wobble position often contains a modified nucleoside, and a correlation exists between the kind of modified nucleoside present in this position and the coding capacity of the tRNA (202) (Fig. 4). Uridines in this position are usually modified (
is the only tRNA in E. coli that has an unmodified U as wobble nucleoside), and they are of two different kinds: the 5-hydroxyuridine derivatives (xo5U34) and the 5-methyl-2-thiouridine derivatives (xm5s2U34). Structural analyses of nucleotides, dinucleotides, and tRNA by nuclear magnetic resonance in solution have revealed that the C2'-endo and C3'-endo forms of the ribose ring of the unmodified Up are almost equally stable, whereas the C2'-endo form of the xo5Up is more stable than the C3'-endo form (3, 272, 327). On the other hand, the xm5s2Up is more rigid and almost exclusively takes the C3'-endo form (3, 272, 327; see review in reference 326). Therefore, the xm5s2 or xo5 modifications regulate the rigidity in the first position of the anticodon in an opposing way, which in turn influences the codon choice. The xo5U derivatives in the wobble position extend the wobble capacity, whereas the xm5s2U derivatives restrict it. This hypothesis is supported by the coding capacities of the corresponding modified tRNAs (see references in references 326 and 327), although some results using mutants defective in the synthesis of mnm5s2U34 (see below) do not support the hypothesis.
tRNA with complementary anticodons can form dimers (80), and this was utilized by Grosjean and collaborators as a useful representation of anticodon-codon interactions (114, 115; reviewed in reference 112). Analysis of the stability and thermodynamic properties of different tRNA-tRNA complexes revealed three major elements that stabilize the interaction: (i) a decreased flexibility of the anticodon induced by the anticodon stem, (ii) the influence of bases outside the anticodon ("the dangling end effect"), and (iii) the presence of modified nucleosides in the anticodon loop, which have a major stabilizing effect due to an enhanced stacking interaction. These three parameters vary in importance in the different tRNA combinations. Such analyses have given important information on how modification may influence the anticodon-codon interaction; e.g., the ms2i6 and t6 modifications of A in position 37 have a strong stabilizing effect mainly due to stacking interaction of the modified adenine to the anticodon triplet (114, 145, 309). In the anticodon-codon interaction, we have to consider at least the identity of the nucleosides in positions 34 to 44 of the tRNA. This conclusion is based both on an artificial model system (tRNA-tRNA interaction) and on the translational efficiency of the tRNA in the living cell (as pointed out by Yarus in his "extended anticodon hypothesis" [325]). Although the extended anticodon is the major determining element in the decoding process, other parts of the tRNA also influence the decoding properties. This aspect is, e.g., demonstrated by the altered decoding properties of the Hirsh suppressor, which has a base substitution in the D-stem of tRNATrp, and by some frameshift suppressors (14, 142a). Therefore, modification outside the anticodon region may also influence the decoding capacity of the tRNA. Furthermore, although the tRNA-tRNA interaction may be a good model system, in vivo the ribosome and the translation factors are active participants in the decoding process.
Completely unmodified tRNA has the same (207) or slightly reduced (252) translational activity in comparison to the native tRNAs. Unmodified tRNAGly and fully modified tRNAGly are similar in their ability to read the different glycine codons (59). However, completely unmodified E. coli tRNAPhe shows considerable defects in the individual rate constants of the translation process (132). Normally this tRNA contains 10 different modified nucleosides including ms2i6A37. It was shown that the presence of these 10 modified nucleosides influences the accuracy of translation by decreasing the rate of peptide synthesis and increasing the rate of rejection of the tRNA in its interaction with noncognate codons. Thus, complete undermodification of a tRNA must be disastrous for the cell. To evaluate the impact of the individual modified nucleosides, it is necessary either to introduce the modification using a purified enzyme, to chemically remove a modification, or to use tRNAs from genetically well-defined mutants deficient in a tRNA modification. All three approaches have provided valuable information on the function of some modified nucleosides.
Function of Modified Nucleosides in Position 32.
In E. coli 
, a s2C is present in position 32. Chemical conversion of s2C32 to C32 changes the structure of the anticodon loop (19). Translation of MS2 RNA in vitro using cell-free extracts from E. coli results in the synthesis of the viral synthetase and the coat protein but also results in several other polypeptides, the latter of which are the results of +1 or –1 frameshifting events (13). Whereas the addition of native (s2C32) in 15-fold excess inhibits the endogenous –1 frameshifting, the addition of s2C32 lacking (C32) does not (19). It was speculated that the native , but not the s2C-lacking counterpart, competes efficiently with the frameshifting tRNA at the frameshifting site (26). If so, the lack of s2C32 may lower the translational efficiency of .
Function of Modified Nucleosides in Position 34.
The cmo5U and mcmo5U modified nucleosides are found in tRNAs specific for valine (GUN), serine (UCN), proline (CCN), threonine (ACN), and alanine (GCN); i.e., they are all part of tRNAs that read families of four codons specifying the same amino acid (Fig. 4). In triplet-dependent binding and in in vitro synthesis of MS2 coat proteins these tRNAs efficiently read codons ending with A, G, or U (149, 187, 192, 213, 253, 283, 284). The xo5 group and the 5'-phosphate interact and thereby stabilize the C2'-endo configuration, which favors the formation of an xo5U34-U and an xo5U34-G base pair. In the C3'-endo configuration, which these uridines are still able to adopt, they form an xo5U34-A base pair (327). Within each codon family the codons ending in U or C are also read by at least another tRNA (Fig. 4). Thus, it is not obvious why these modifications are present in this group of tRNAs. However, an Aro– mutant of E. coli, which completely lacks xo5U modifications and has an unmodified U in the corresponding tRNAs (24, 126), grows 20% slower than Aro+ cells in rich medium. Therefore, under such conditions the presence of cmo5U is important (G. R. Björk, unpublished observation).
Model experiments, as discussed above, have shown that the xm5s2U34 nucleosides restrict an intrinsic wobble capacity of the uridine. Results from triplet-dependent binding and in vitro protein synthesis also support this model, since these modified nucleosides primarily recognize A as the third letter of the codon (180, 262). However, analyses in vivo using mutants defective in the synthesis of these kinds of modified nucleosides have not always given results in support of the model. The ochre suppressor 
(supG or supL) reads UAA and UAG nonsense codons and has mnm5s2U34. This tRNA contains cmnm5s2U34, nm5s2U34, and s2U34 in trmC1, trmC2, and trmE mutants, respectively (82, 123). Each mutation reduces the efficiency of the suppressor tRNA, but each affects the reading of UAG more than the reading of UAA. Furthermore, lack of mnm5 modification, as in the trmE mutant, also affects the binding of tRNALys to AAG more than the binding to AAA (82). Thus, the modification at the 5 position increases the binding towards G rather than restricting it, according to the aforementioned theory. Therefore, the primary function of the mnm5 side chain may not be to impose the selective reading towards A, but to increase the efficiency of the anticodon-codon interaction to both kinds of codons and preferentially to G-ending codons. If so, the restrictive reading of tRNALys is mainly imposed by the s2 group. tRNAGlu, which normally has mnm5s2U34, lacks the s2 group when purified from cysteine-starved cells (4). Such undermodified tRNA binds in vitro much better to GAG than to GAA, whereas the reverse is true for the normal 
(4). These results support the hypothesis that the s2 group of mnm5s2U34 imposes a restrictive wobbling towards G. However, the restrictive coding capacities of 
, which contains cmnm5Um34, or of 
, which contains mnm5U34 (250, 282a) (see Fig. 4), must be imposed by these modifications, since these modified nucleosides do not contain the s2 group.
The selD mutant (denoted selA1 in reference 168) is unable to incorporate Se into the tRNA. In such a mutant, an ochre suppressor 
derivative encoded by the supG gene contains only mnm5s2U34, whereas in the wild-type selD +, 40% of the wobble nucleoside is mnm5Se2U34. The selD1 mutation imposes a twofold reduction of the to read UAG but does not affect the reading of UAA (168). Apparently, the presence of selenium instead of sulfur at position 2 of uridine improves the ability to wobble. This is consistent with results obtained in vitro in triplet binding experiments (319). and containing an s2 group perferentially bind to A-ending codons, whereas for the Se2-containing tRNAs this preference is either reversed (tRNALys) or diminished (tRNAGlu). Therefore, replacement of sulfur by selenium of the mnm5s2U34 increases the wobble interaction with G (319).
The elongator Met-
is the only tRNA in E. coli that contains ac4C, which is present in the wobble position. This suggests a unique function of the acetyl group present in this tRNA. Upon removal of the ac4 group by bisulfite, the tRNA gains the ability to misread the isoleucine codon AUA (280) and also becomes more efficient in recognizing the cognate codon AUG. Note that the AUA (Ile) codon is normally read by a minor tRNAIle, which has the modified nucleoside k2C34 (L34) in the wobble position (see Fig. 4). Thus, the ac4 modification in the CAU anticodon of 
decreases the efficiency to read both the complementary AUG codon and the noncomplementary AUA codon. The ability for C to read A most likely requires a specific conformation of the tRNAs, since most amber suppressors, which normally have C34, are unable to read the ochre codon UAA. Thus, the function of the ac4 modification appears primarily to prevent misreading of AUA (Ile) and this is achieved by a slight reduction in the ability to read the cognate AUG (Met) codon by the 
.
The AUA (Ile) codon is normally read by a minor species of 
(131). This tRNA has lysidine (k2C34, also abbreviated L34) in the wobble position (191) and recognizes only AUA (131). Thus, the modification C34 to k2C34 alters the wobble nucleoside so that A is recognized instead of G. This is not a wobble but a complete conversion of a base-pairing specificity (190). As discussed above, this modification also inhibits the misacylation of methionine. Therefore, a single posttranscriptional modification converts both the aminoacylation and the codon specificity.
The tgt mutant completely lacks Q in tRNAs specific for Tyr, His, Asn, and Asp (203). The his operon is regulated by an attenuation mechanism, which is sensitive to the charging and translational efficiency of tRNAHis (153). A reduced rate of cognate decoding of the seven his (CAU) codons in the attenuator results in a derepression of the his operon. No difference in the expression of the his operon was observed in tgt + and tgt cells, suggesting that the Q-deficient tRNAHis is as efficient as the wild-type tRNA in translating the His codons in the leader region of the his attenuator. However, the binding efficiency of G34-tRNATyr to triplet programmed ribosomes is twofold less than that of Q34-tRNATyr, although the preference to recognize UAU over UAC is the same in both Q34- and G34-containing tRNATyr (203). Thus, whereas Q34 does not influence the decoding efficiency of tRNAHis, the efficiency of E. coli tRNATyr to bind to ribosomes is reduced twofold without change in the codon choice. This suggests that the influence of Q34 on individual tRNA species may be different. In other systems, the Q has also been shown to influence the codon choice (182). The tgt mutant is also able to induce a weak suppression of some amber mutations, suggesting that lack of Q34 may induce some missense errors (96), as has also been observed in vitro (22, 23). However, Q34 deficiency does not provoke a major change in the growth rate of the tgt mutant (203).
In summary, modifications in position 34 may increase (cmo5U and mcmo5U) or decrease (mnm5s2U, mnm5Um, mnm5U) the ability to wobble, and results from model experiments in vitro of tRNA in solution have presented a molecular explanation for such a pairing ability. Although in vivo analyses have shown that the mnm5s2U34 wobble nucleoside is optimal for an efficient reading of the tRNA, they have so far not shown that the mnm5 side chain imposes any restrictive wobble capacity as suggested by the aforementioned hypothesis. On the other hand, the s2 group may be the major contributing factor in the postulated restrictiveness of mnm5s2U, although for some tRNAs (Leu and Arg) ribose methylation and/or the mnm5 side chain must be the restrictive agent(s). The acetylation of C (ac4C) prevents misreading at the expense of a reduced ability to read the cognate codon, and the lysylation of C (k2C) completely changes the coding capacity of C from pairing with G to pairing with A. The hypermodified Q may or may not increase the efficiency of reading the cognate codon, but may mediate a weak increase in fidelity.
Function of Modified Nucleosides in Position 37.
Figure 4 shows that tRNAs reading codons starting with U or A have ms2i6A37 or ms2io6A37 and t6A or m6t6A in position 37, respectively. The weak A36-U or U36-A interactions between position 36 of the anticodon and the first position of the codon may be stabilized by these hypermodifications and prevent first-position errors (74, 154, 201, 309). If so, codons starting with C or G, which results in a strong G-C base pairing between position 36 of the anticodon and the first position of the codon, would not require any stabilizing modification in the nearby position 37, and indeed, unmodified A or methylated A or G is found in position 37 of such subsets of tRNAs (Fig. 4).
Measuring the stability of complexes between tRNAs with complementary anticodons, it was found that the t6A modification stabilizes anticodon-codon interaction, most probably by increased stacking interaction (309). Furthermore, a mutant, mtaA1, most likely lacking the methyl group of m6t6A in the only tRNA (
) that contains this modified nucleoside, shows a decreased efficiency to read the cognate codon ACC (Qian and Björk, unpublished observation). Therefore, these results support the suggestion that the t6 modification, which is present in this subset of tRNA from all organisms, improves the efficiency of the tRNA. So far, no results have been obtained addressing the question of whether or not the t6 modification also improves first-position fidelity.
The hypermodified nucleosides ms2io6A37 (S. typhimurium) and ms2i6A37 (E. coli) are present in tRNAs that read codons starting with U (the only exceptions are 
, which has A37, and 
, which has i6A37). Consequently, these modified nucleosides are present in amber suppressor tRNAs, which have made it possible to determine the efficiency of such tRNAs in vivo only differing in the degree of this modification. The influence of the codon context can also be analyzed by placing the amber codon in different contexts. Such analyses have revealed that a complete lack of the modification, as in a miaA mutant, results not only in a dramatic reduction (up to 99%) of the efficiency of the suppressor tRNA, but also induces an increased codon context sensitivity of the unmodified tRNA (31, 34, 87, 225, 300). The ms2io6 modification specifically counteracts an unfavorable nucleotide on the 3' side of the codon (87). Furthermore, the lack of ms2i6 modification also reduces the efficiency of the tRNA in vitro (71, 101, 300). The major effect on the efficiency of the tRNA is imposed by the i6 group of ms2i6A37, since a miaB mutation, which results in a tRNA having i6A37 instead of ms2io6A37, has much less effect than a mutation in the miaA gene (125). This result is also consistent with results obtained in vitro (45, 101). The hydroxyl group present in S. typhimurium tRNA has no effect on the efficiency of tRNATyr (222). Therefore, both the i6 and the ms2 groups contribute to the function of ms2io6A37, whereas hydroxylation, which only occurs in S. typhimurium, does not influence the anticodon-codon interaction. It was suggested that the ms2i6 modification at position 37 of a tRNA, making an A36-U base pair with the first nucleoside (U) in the codon, would stabilize this base pairing and inhibit the wobble capacity and thus reduce a fatal first-position misreading (74, 154, 201). This hypothesis is supported by an in vitro experiment in which it was shown that the presence of the ms2i6 modification reduces a first-position error (132, 317). Supporting evidence has also been obtained in vivo (34). On the other hand, third-position errors are increased by the presence of the modification both in vivo (31, 34, 225) and in vitro (71, 300), most likely by decreased proofreading (31, 71). Thus, when a mismatch occurs in the wobble position, the ms2i6 modification increases such near-cognate misreading by decreased proofreading, whereas it is neutral or decreases the first-position misreading by noncognate tRNAs. These apparently conflicting results are, in fact, consistent with a hypothesis that a modification which increases third-position wobble would decrease first-position errors (200a). Thus, ms2i6A37 enhances the activity of the tRNA, makes it less sensitive to the codon context, and increases and decreases the first- and third-position fidelity, respectively. The physiological consequences of m2i6 deficiency result in a drastic reduction (up to 50%) of the growth rate and of the polypeptide chain elongation rate (72, 86, 184). Furthermore, such undermodification alters the expression of several operons and induces a pleiotropic effect on cell physiology (86). Thus, ms2i6A37 has a profound influence on the physiology of the bacterial cell and plays a fundamental role in the efficiency and fidelity of translation.
tRNA from all organisms reading codons of the type CUN (Leu), CCN (Pro), and CGG (Arg) (Fig. 4) contains m1G37, which suggests that this modified nucleoside was present already in the tRNA of the progenitor (25). The function of m1G37 may, therefore, be the same irrespective of the origin of the tRNA. A mutant (trmD3) of S. typhimurium defective in the formation of m1G37 grows 30% more slowly than the wild type in glucose minimal medium, and the chain elongation rate is reduced. This suggests an effect by m1G37 on the efficiency of tRNA in the cognate interaction. The translation elongation cycle consists of several steps, of which the first is the primary selection of the ternary complex. Lack of m1G37 affects the efficiency with which the ternary complex binds to the A-site on the ribosome (125), which at least partly explains the reduced elongation rate of the m1G37-lacking tRNA.
Lack of m1G37 also induces an aberrant anticodon-codon interaction, which results in a capacity to suppress frameshift mutations of the type CCC-C or CCC-U. (CCC is in the zero frame and the next nucleoside is the first base in the downstream codon. m1G37 deficiency induces the ability to formally read this base, which results in a +1 frameshift.) A model was proposed (Fig. 8) in which the unmodified G37 forms a Watson-Crick base pair with the first base in the zero frame codon, thus creating a quadruplet anticodon-codon interaction (30). The addition of a methyl group at position 1 of Gua prevents the formation of one of the three hydrogen bonds that are found between a G and a C in a Watson-Crick base pair (Fig. 8). Thus, the presence of the methyl group prevents a possible interaction between m1G37 and a C in the mRNA. The model was verified for frameshifting at two proline sites (CCC-U/A) by sequencing the resulting peptide (127). Also at other proline sites and at two leucine sites (CCU-C/U) a quadruplet translocation most likely occurs according to the model if m1G37 is absent (Fig. 8). Thus, the presence of m1G37 affects the cognate anticodon-codon interaction by increasing the aminoacyl-tRNA selection and the polypeptide chain elongation rate. Furthermore, it prevents frameshifting by inhibiting a Watson-Crick base pairing with a C in the mRNA. This role of m1G37 to prevent frameshifting may be the same in tRNAs from all organisms and may have evolved early, since m1G37 may have been present in the tRNA from the progenitor.
In summary, ms2i(o)6A37 increases the efficiency of the tRNA and make it less codon context sensitive. The presence of the ms2i(o)6 modification increases third-position errors whereas first-position errors are reduced. The presence of m1G37 maintains the reading frame and increases the efficiency in the cognate interaction. Lack of these kinds of modifications has a profound influence on the physiology of the cell and induces a substantial decrease in the growth rate.
Function of ψ in the Anticodon Region.
A mutation in the hisT gene results in a deficiency of ψ in the anticodon loop (position 38) and stem (position 39 and 40) (Fig. 2) in 17 of the 40 tRNA species sequenced, among them tRNAHis (278). The histidine leader mRNA contains seven consecutive histidine codons that are read inefficiently by tRNAHis lacking ψ38,39. This results in a derepressed his operon (153). The growth rate of the hisT mutant is reduced by 30% in glucose minimal medium, as is the polypeptide chain elongation rate (218). Comparison of the effect of ψ deficiency of two amber suppressors, supF and supE, which have ψ at position 39 alone or at both positions 38 and 39, respectively, suggests that the ψ at position 38, i.e., in the anticodon loop, exerts in a codon context-independent manner a stronger influence on the activity of the tRNA than ψ in position 39 (33, 124). One of the steps affected by ψ is the aminoacyl-tRNA selection step (125). Also, misincorporation by a near-cognate tRNA is correlated by ψ modification at position 38 (219). Thus, ψ in the anticodon region has a strong impact on the activity of the tRNA, and although ψ in the stem increases the activity, this effect is less than the one exerted by ψ in the loop (position 38). The ψ modification creates an extra imino hydrogen at the nitrogen that is engaged in the glycosyl bond of unmodified U (see Fig. 1). This imino group has the potential to form a hydrogen-water bridge to an oxygen of the ψ5'-phosphate, resulting in a more stable structure (70). Since ψ in position 38 is not involved in Watson-Crick base pairing, the ψ38 modification may exert its stronger influence on the activity of the tRNA by creating water or magnesium bridges within the anticodon loop. Such bridges may stabilize and adapt the anticodon to interact with the mRNA in a more efficient way.
Function of Modified Nucleosides in Other Positions than the Anticodon Region.
Little knowledge of the function of modified nucleosides outside the anticodon region has been obtained. The reason may be that their effects on the anticodon-codon interaction are minor and that the methods employed are too insensitive to reveal certain functions of these modified nucleosides. A nuvA mutant lacking s4U in the tRNA grows normally (292), but this nucleoside is directly involved in photoprotection. Only a minor reduction (4%) of growth rate was observed for a mutant (trmA) completely lacking m5U54 in its tRNA; however, such undermodified tRNA had lower stability, increased error level, and decreased A-site binding in vitro (69, 161). Although the presence of m5U54 in the tRNA is not essential for cellular growth, the structural gene (trmA) for the tRNA(m5U54)methyltransferase is (223). It was therefore suggested that the TrmA peptide has two functions: one catalyzes the formation of m5U54 in tRNA, and the other activity is involved in an unknown but essential function. The unknown function may be related to the observation that 50% of tRNA(m5U54)methyltransferase is covalently bound to 16S rRNA and undermodified tRNA (120). This enzyme also has a low capacity to form m5U in position 788 in 16S rRNA (116). However, whether these observations are related is not known. Nevertheless, the trmA gene is so far the only tRNA modification gene shown to be essential.
This work was supported by grants from the Swedish Cancer Society (project 680) and the Swedish Natural Science Research Council (project B-BU 2930). The critical reading of the manuscript and stimulating discussions with B. Esberg, C. Gustafsson, T. Hagervall, J.-N. Li, O. Olafsson, Q. Qian, L. Taraseiciene, J. Urbonavicius, and H. Wolf-Watz are gratefully acknowledged. I am also indebted to J. McCloskey, Salt Lake City, Utah, and J. Ofengand, Nutley, N.J., for unpublished results.
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