Translational Initiation
Chapter
59
DAVID E. DRAPER
Initiation is usually the rate-limiting step of translation, and its efficiency can vary by several orders of magnitude. It is therefore a major determinant of the overall expression level for a gene and is also a target for a variety of translational control mechanisms. Although the outlines of translational initiation have been evident for 25 years, a quantitative understanding of the factors determining the overall initiation rate has been very slow in coming. There have been advances in two areas in recent years that put translational initiation on a reasonably secure footing. One is a preequilibrium model that is able to relate mRNA sequence and secondary structure to translational efficiency and to provide some understanding of initiation factor function; though the model is not overly detailed, it does provide a solid framework for interpreting a great deal of in vitro and in vivo rate measurements. The other area is translational initiation at internal sites of multicistronic operons, which in many instances is strongly coupled to translation elsewhere in the mRNA and is exploited by the cell to regulate stoichiometric ratios of specific proteins.
Translational initiation is the sequence of events leading up to formation of the first peptide bond. The components of the initiation complex have been identified for many years: the ribosome, an mRNA, fMet-
, and three initiation factors (IF1, IF2, and IF3). During initiation at least one GTP is required as well. The order in which these components assemble into an initiation complex has been the subject of considerable study, reviewed most recently by Gualerzi and Pon (18). The best summary at present is that a complex of a 30S subunit with each of the initiation factors binds mRNA and fMet- in random order. This "preinitiation" complex then undergoes a rate-limiting unimolecular rearrangement and subsequently binds a 50S subunit with the concomitant expulsion of IF1 and IF3. The last step is the hydrolysis of GTP to GDP and release of IF2. The final complex is exceedingly stable, and for practical purposes its formation may be considered irreversible.
The only variable component in the initiation scheme is the mRNA; both the specificity of initiation for selected start sites and the several-orders-of-magnitude variation in translational efficiency of different start sites must be encoded in the mRNA. Early RNase protection studies indicated that the ribosome contacts at most ∼30 nucleotides (nt) surrounding the initiation codon (57). Later statistical studies have come to the same conclusion: in alignments of hundreds of known translational start sites by their initiation codons, the region deviating from random sequences extends –20 nt upstream of the initiator and +13 nt downstream (48), for a total of 34 nt. (The A of the AUG codon is numbered 0.)
Within the translational initiation region are two obvious sequence features. The first is the initiation codon itself, which is most often AUG; GUG is used in a few percent of start sites (58). Other codons are very rare. UUG is used to initiate ribosomal protein S20, and AUU is the initiator for infC (IF3). Both of these genes are autogenously regulated, and the unusual codon is required for the regulation in each case (4, 38). The second feature is the Shine-Dalgarno (SD) sequence, which is located upstream of the initiation codon and is complementary to a sequence at the 3' terminus of 16S rRNA. That this sequence is involved in initiation site selection has been unequivocally demonstrated by in vivo production of ribosomes containing an altered anti-SD sequence in their 16S rRNAs. These "specialized" ribosomes synthesize protein only from mRNAs containing the complementary changes in the SD sequence (24).
The optimum spacing between the SD sequence and the initiation codon places C1535 of the 16S rRNA about 8 nt away from the A of the initiation codon. The ribosomal machinery does not demand a precise distance; only when the spacing drops below 4 or above ≈14 does the translation rate decrease by more than 10-fold (15, 42). The importance of each of these three aspects of a ribosome binding site sequence (initiation codon, SD sequence, and the spacing between them) has been shown by measurements of β-galactosidase activity in strains carrying a series of related binding site sequences (42).
The statistical preference for the remainder of the ∼30-base initiation region is merely for A-richness and not for a specific sequence (15, 58). Though this requirement has been less extensively documented, at least two experimental studies have shown that A-richness is a significant determinant of ribosome binding sites (13, 26). This purpose of this sequence bias may be in part to prevent secondary structure formation.
There have been a number of suggestions that additional mRNA sequence elements with complementarity to other parts of the 16S rRNA may function in the same way as the SD sequence and serve as enhancers for efficient translation of certain classes of mRNAs. These suggestions have been based on either statistical analysis (29, 59) or observation of mutations increasing gene expression (14, 36). The former studies have not yet been subjected to any kind of experimental verification. Given the large size of the 16S rRNA and the large number of ribosome binding site sequences in the literature, it is expected that some very striking but completely coincidental complementarities will be detected. It is probably best to maintain some scepticism regarding translational enhancer sequences until more thorough experimental confirmation (e.g., compensatory mutations in the mRNA and rRNA) is presented.
The 30S subunit, mRNA, and fMet- form a very stable ternary complex in the absence of initiation factors. The development of a simple assay for this complex has stimulated a number of studies of the roles of mRNA sequence features and initiation factors in promoting correct and efficient initiation. The assay is the premature termination of a reverse transcriptase reaction initiated with a DNA primer hybridized downstream of the initiation site. The termination site is almost invariably found 15 nt into the coding region and presumably reflects a steric blocking of transcriptase activity by the "edge" of the tightly bound ribosome, hence the popular term "toeprint" for the truncated transcripts.
Since the ternary complex has a half-life of a number of hours, the intensity of a toeprint band reflects the association rate of the complex (55). In quantitative studies with a number of ribosome binding site sequences, this rate is enhanced by (i) AUG initiation codon over GUG or AUA, (ii) lengthened SD sequence, and (iii) an optimum spacing between the SD sequence and the initiation codon (23). The parallel between these requirements for toeprints and those for efficient translation in vivo (42) argues that a genuine initiation complex is being observed in these experiments.
30S subunits alone bind mRNA or synthetic polymers, but the affinity increases by an order of magnitude if an SD sequence is present (5). The toeprint assay is able to detect the specific 30S-mRNA complex under suitable conditions (22). The position of the toeprint signal (and presumably the ribosome edge) shows little change upon formation of the ternary complex (41). Taken together with the observation that the SD pairing stimulates but is not essential for translation, these observations argue that the initial selection of a ribosome binding site (and not subsequent steps) is strongly influenced by mRNA-16S rRNA base pairing.
A toeprint complex formed in the absence of initiation factors is defective in that there is no specificity for initiator . Not only are tRNAMet and not distinguished, substitution of a tRNA specifying the second codon shifts the toeprint signal 3 nt downstream (21). IF3 increases the specificity for binding at the initiation codon and will even promote the binding of a small hairpin duplicating the anticodon stem and loop in competition with other tRNAs (21). (IF3 discriminates against complexes with long SD-to-initiation-codon spacings as well [23].) IF2 also increases the specificity of the initiation complex for , but only if fMet- is used (21). A role for IF1 becomes apparent only when 70S ribosomes are used instead of 30S subunits. Neither IF2 and IF3 together nor IF1 alone favors fMet- with 70S subunits, but the combination of all three factors promotes fMet- -containing complexes (23).
A number of other studies indicate that the initiation factors do not affect mRNA-30S subunit binding affinity, but do alter the kinetics of initiation complex formation and dissociation, the positioning of the mRNA within the subunit, and selectivity for initiator tRNA (5, 6, 19; reviewed in reference 18).
Kinetic schemes for translational initiation have been published by several laboratories (12, 18, 42); all are similar and parallel a basic scheme proposed by McClure for transcriptional initiation (32). In the transcriptional scheme, initial binding of polymerase to the promoter is followed by an irreversible conformational rearrangement (isomerization) to the open complex and transcription. To transpose this two-step mechanism to translation, an initial binding between 30S subunits and mRNA (the binary complex) is presumed to take place, stabilized primarily by the SD interaction; fMet- may be present but does not participate in this initial event. This "standby" or preinitiation complex then undergoes conformational rearrangement(s) to give the stable ternary complex (Fig. 1).
In a two-step scheme of this sort, there are two limiting cases of interest. If isomerization is fast compared to the rate of 30S-mRNA dissociation, then the overall binding affinity is unimportant; every subunit that associates with mRNA is engaged in translation without a chance to dissociate. The translation rate is then proportional to k 30S[30S] and is said to be binding limited. ([30S] is the concentration of free subunits.) When the isomerization step is slow, equilibrium is established in the first step, and the occupancy of the initiation site depends on the subunit-mRNA binding affinity. In this case, the translation rate is proportional to K 30S[30S]k isom and is isomerization limited. (K 30S is the ratio k 30S/k –30S .)
Unfortunately, a complete set of parameters from equilibrium and steady-state kinetic measurements are not available. K 30S is on the order of 107 to 108 M–1 (5, 12). Estimates of the isomerization rate are more difficult to make from available data; a kinetic study of tRNA binding suggested that it is on the order of 0.1 s–1 (63), roughly consistent with the observation that an efficient mRNA initiates translation every ∼3 s (27). The crucial piece of information that decides whether equilibrium is established in the first step is the relative magnitude of isomerization (k isom) and subunit dissociation (k –30S); this has not been directly determined. However, a large number of in vivo rate measurements make sense if (in most mRNAs) k –30S is fast and the translation rate is proportional to K 30S[30S]k isom .
The principal argument for subunit binding coming to equilibrium is as follows. Experimental measurements show that the bimolecular subunit-mRNA affinity is strengthened by the SD base pairing (5), and in vivo experiments also show that translational efficiency tends to increase with the extent of SD complementarity (42). If translation is binding limited, then a strengthened SD interaction must have increased the association rate; if it is isomerization limited, the SD interaction need only have strengthened K 30S . The rate of forming a double helix is independent of the stability of the helix; it is the dissociation rate that slows with increasing stability. Thus an effect of the SD on K 30S (but not k 30S) is implied. Additional observations consistent with isomerization-limited kinetics are discussed below in relation to secondary structure effects on translation rates.
How might other features of an initiation site affect the isomerization-limited mechanism? Ringquist et al. (42) have suggested that the spacing between the SD sequence and initiation codon primarily affects the isomerization rate. Direct kinetic measurements to support this are lacking, but the hypothesis is consistent with in vivo translation rates of a series of homologous mRNAs. These same workers also complicate the Fig. 1 scheme by including dissociation of the isomerized complex (k –isom) in competition with translation (k trans); they attribute the (generally) decreased efficiency of GUG or UUG initiators to an increase in k –isom .
The toeprint assay has come into common use as a method for detecting ribosome binding sites, and it is frequently assumed that toeprint "strength" reflects the in vivo efficiency of the start site. Although a rough correlation has been seen in some instances (23, 42), the assumption has not been extensively tested. Based on the observations that (i) the ternary toeprint complex is extremely stable, (ii) the rate of toeprint appearance is slow, on the order of 1 to 10 min, and (iii) the toeprint signal increases with increasing SD complementarity, it is very likely that the same isomerization-limited kinetics apply to the toeprint assay as to in vivo translation. Since initiation factors do not affect the subunit-mRNA equilibrium (5), the principal kinetic difference between the toeprint assay and in vivo kinetics is probably a much slower isomerization step for the toeprint. As long as both the toeprint and in vivo translation are isomerization limited, relative toeprint strengths will be a good measure of relative in vivo translation rates. The cases in which toeprint intensity is a poor predictor of in vivo efficiency tend to be mRNAs in which secondary or tertiary structure has a large influence on the translation rate (54); it may be that these exceptions do not follow isomerization-limited kinetics.
Accessibility of the SD sequence and initiation codon for base pairing is needed for initiation complex formation, and it was unequivocally demonstrated by Hall et al. (20) that subtle changes in secondary structure modulate the rate of translation in vivo. If initiating ribosomes must compete with mRNA secondary structure for access to the initiation region, then the initiation scheme should be amended with the competing equilibrium Kf shown in Fig. 1. Making the further assumption that both the initial 30S-mRNA complex and the secondary structure formation come to equilibrium, the relative rate of translation then becomes dependent on the free energy of the secondary structure, and the proportionality
should hold, where Kf is the equilibrium constant for folding the secondary structure and K 30S[30S] is the product of the subunit-mRNA binding constant and concentration of free subunits (9). The in vivo translation rates for a series of mutations altering the stability of a hairpin containing the MS2 coat protein gene follow this predicted proportionality (9).
The above equation predicts that a plot of ln(translation rate) as a function of Δ Gf (recall that Δ Gf = -RTlnKf) will have two limiting slopes. When the secondary structure is stable (Δ Gf is very negative), decreasing the structure stability by 1 kcal/mol should increase the translation rate 5.1-fold (at 37°C). But if the secondary structure is weak (Kf < K 30S[30S]), the binding energy of the ribosome will easily melt the structure and drive translation to the maximum possible rate. This shift to structure-independent translation rates as mRNA secondary structure is weakened provides a direct measure of 30S subunit-mRNA binding free energy in vivo. De Smit and van Duin (10) systematically varied the stability of a hairpin in the initiation region of the MS2 coat protein mRNA and found that the binding free energy is about –11 kcal/mol, corresponding to K ≈ 5 × 107 M–1 . This is in accord with in vitro measurements (5).
By the Fig. 1 initiation scheme, increasing the SD complementarity should make a more stable subunit-mRNA complex that will compete more effectively with mRNA secondary structure. Precisely this effect is seen: either increasing or decreasing the number of potential SD base pairs altered the apparent binding free energy by roughly the amount predicted for the change in mRNA-rRNA stability (11). An unexpected result of these experiments with different SD complementarities is that the maximum translational efficiency, reached for those mRNAs with sufficiently unstable structure, was independent of the SD sequence length. This suggests that the in vivo level of free ribosomes is sufficient to saturate unstructured mRNAs. To a first approximation, the wide range of observed translation rates is attributable to the balance between the extent of SD interaction and the stability of mRNA secondary structure.
What about mRNA structure within the ribosome binding site but not including the SD or initiator sequences? Toeprints with mRNAs containing stable hairpins just before or after the initiation codon suggest that the mRNA "track" is roomy enough to accommodate secondary structure within the initiation complex (41). The free energy of binding need only drive the denaturation of enough structure to find the two critical features, i.e., the SD and initiator sequences, leaving denaturation of any structure within the coding region for the elongating ribosome to deal with.
Many Escherichia coli mRNAs are multicistronic, which introduces two additional complications to translational initiation. First, translation of a specific gene may become dependent on translation taking place elsewhere in the mRNA; the phenomenon is referred to as ribosome binding site "induction" in an excellent review of the topic (8). Second, translation of downstream cistrons may be initiated by ribosomes that have finished translation of an upstream gene, but have not yet dissociated from the mRNA. This reinitiation phenomenon can potentially increase the efficiency of a ribosome binding site from what would be observed for the same site in a monocistronic mRNA. Both inducible initiation sites and reinitiation are used by E. coli to regulate the stoichiometric ratios of products from multicistronic operons and are considered in turn below.
The first observation of initiation site induction was in the MS2 phage, in which the coat protein gene must be translated before expression of the replicase is observed (60). The fact that denaturation of the phage RNA weakened coupling between the two genes suggested that RNA structure is responsible. This was nicely confirmed by site-directed mutations that either disrupted or restored Watson-Crick pairing within a potential 7-bp helix; coupling was either abolished or restored in the respective mutants (61). Thus a sequence at codons 31–32 of the coat gene is linked to a sequence at –17 to –23 relative to the replicase initiation codon; there are a remarkable 305 nucleotides separating the two sites. It is also noteworthy that the helix does not involve either the SD or initiation codon, both of which are contained within a weak hairpin structure that is the target for translational repression by the coat protein itself. Coaxial stacking of the long-range pairing with the hairpin could stabilize the latter by roughly 1 kcal/mol, sufficient to produce the coupling effect if the translation rate is limited by the hairpin stability (see above discussion of secondary structure). Translational control in this phage is evidently effected by a set of RNA structures energetically poised so that small changes in stability have substantial effects on translational efficiency.
Another example of long-range coupling is the activation of rplL translation when ribosomes traverse the first 40 nt of rplJ, more than 500 nt upstream (39). The coupling helps maintain the synthesis of the downstream rplJ product, ribosomal protein L7/L12, at a fourfold higher rate than rplJ (L10), which is the needed stoichiometric ratio for the two proteins in the ribosome.
Over a shorter-sequence range, existence of a specific secondary structure that guarantees strong translational coupling between the MS2 phage coat protein and the downstream lysis gene has been established. The coat gene termination codon is actually downstream of the lysis gene initiation codon, and in a different reading frame; termination at the coat stop codon and not merely elongation through structure containing the initiation site is required to observe lysis protein synthesis (3). Mutations that increase the stability of an extended stem structure surrounding the lysis gene initiation site suppress its synthesis, while mutations weakening the structure uncouple lysis gene translation from that of the upstream coat protein (46). Coupling can be restored in mutants with increased stem stability by moving the coat termination site closer to the lysis initiation site. In this case, translational initiation levels depend on a balance between secondary structure stability and initiation codon position.
Reinitiation of translation was first observed in genetic studies of nonsense mutations in lacZ and T4 rIIB (16, 43) and has been extensively studied in these and other systems (34, 37, 50). The original supposition that 30S subunits might remain bound to the mRNA for some time after termination and be able to reinitiate at nearby start sites without dissociation has proved essentially correct. In the cases of restart sites that occur within a few nucleotides of the termination codon of the upstream gene, mRNA secondary structure has presumably been denatured by the terminating ribosome and the mRNA primary sequence is the principal determinant of restart efficiency. In general, the same sequence requirements apply to restart sites as to independent initiation sites: AUG is better than GUG or UUG, and longer SD sequences are better (31). However, the requirements appear much less stringent than for independent initiation. Deletion of the SD region reduced translation 10-fold in one instance, but the level was still detectable (53). In an extreme case, translation of the fd phage gene VII is observed only when an upstream gene is present (in the phage, gene V), and not when the ribosome binding site is at the beginning of a message. Although the normal AUG initiator is used, the sequence has only a two-base potential SD complementarity, and the overall region is considerably more G-C rich than an average initiation region (26). Mutations which lengthen the SD complementarity or alter G’s and C’s to A’s or U’s throughout the site additively increase its efficiency to approach that of a normal independent initiation region (26).
The relaxed sequence requirements for reinitiation are understandable if termination delivers a steady supply of 30S subunits to the restart site, so that the site need not have a large subunit binding affinity to compete for subunits from the cellular pool. The efficiency of reinitiation should therefore depend on the distance of the restart initiation codon from the termination codon; over a long enough distance, dissociation of the subunit must eventually be favored. A systematic investigation of the distance dependence has provided convincing evidence that a subunit can "slide" along the mRNA for distances up to 40 nt in either direction from the termination codon; the first suitable initiation codon encountered is the one used (1). It should be noted that elongating ribosomes consume GTP and thus can denature rather stable secondary structures. After termination this energy is no longer available, with two consequences: sliding should be more sensitive to secondary structure, and there should not be any directionality to the movement. These predictions are confirmed by experiment, although diffusion in the 5' direction is somewhat less efficient, probably because of collisions with elongating ribosomes (1).
The above discussions of induction and reinitiation suggest that there are two strategies that could be used to strongly couple translation of two adjacent genes on an mRNA, so that independent initiation of the downstream gene does not occur. The first is to simply use a defective sequence that cannot capture ribosomes on its own, but is able to initiate if supplied with ribosomes from a nearby terminator. This is the strategy used by the fd gene VII (25). It is useful when the downstream gene product is needed at a fraction of the level of the upstream product, but a stoichiometric ratio of the two genes is probably not possible because the strategy depends on an inherently poor initiation region. The alternative is to combine induction and reinitiation, so that the downstream site is masked by secondary structure and accessible only to ribosomes that have terminated translation of the upstream gene. This strategy is evidently used extensively among the ribosomal protein operons of E. coli, in which all but one of the proteins are required in equimolar ratios. (The one exception of a ribosomal protein present at higher levels, L7/L12, was already mentioned above.) The synthesis of most ribosomal proteins in E. coli takes place from several multicistronic operons, all of which utilize a feedback translational control mechanism (35). A single protein in each operon acts as a translational repressor that binds at a single target site in its own mRNA, repressing synthesis of all the ribosomal proteins in the operon. The regulation has been demonstrated by in vivo and in vitro experiments and requires that translation of all the ribosomal proteins in an operon be tightly coupled.
Translational coupling has been demonstrated in two cases that have been examined. The L11 termination codon is three bases from the L1 initiation codon, and tight coupling between these genes is seen (2). The coupling is almost certainly promoted by stable secondary structures near the C terminus of the L11 gene and within a region genetically defined to be required for coupling, though the details of the base pairing have not been established (52). A fairly long sequence is present between the S12 and S7 genes: 96 nt. Coupling between these is nevertheless 80 to 90% efficient (44), and "structure mapping" and compensatory mutations provide evidence for an extensive secondary structure that brings the S12 termination codon into juxtaposition with the S7 initiation codon (45). A ribosome at the S12 termination site would probably denature a sufficient amount of the intercistronic secondary structure to expose the S7 SD sequence. Given the ability of initiating ribosomes to incorporate fairly large secondary structures within the mRNA track (41), it may be possible for a terminated ribosome to "jump" directly to the S7 initiation site without denaturing the intervening secondary structure. The same secondary structure is also the target site at which S7 binds as a translational repressor. Only the coupled translation is sensitive to S7 binding, and not independent initiation (44), perhaps because the protein disrupts the structure that promotes translational coupling.
There are other situations in which the cell uses the presence or absence of secondary structure to regulate translation of a gene. For instance, read-through transcripts of transposons are translated poorly, compared to the expression level seen with normal transcripts. The difference is attributable to secondary structure that forms only in the longer transcript (49). A similar situation occurs in the expression of T4 phage lysozyme; translation of early promoter transcripts is blocked by a secondary structure that is not present in shorter, late promoter transcripts (28, 33).
The above discussion indicates ways that the cell may balance the SD sequence length, secondary structure within the ribosome binding site, and location of termination codons to adjust rates of protein synthesis. This level of regulation is built into the mRNA sequence. It is also feasible for proteins or other RNAs to bind to an mRNA and alter its translation efficiency. The targeting of a structure between the S12 and S7 genes by S7 was mentioned above; there are many other examples from E. coli and its phages of translational control exerted by regulatory proteins and RNAs.
How might a protein repress translation by binding mRNA? The obvious mechanism is for the protein to increase the stability of a secondary structure containing the SD sequence or initiation codon. This is undoubtedly the way in which the R17 coat protein represses synthesis of the replicase gene. The replicase initiation site is contained within a small hairpin that forms with Δ G = –7.7 kcal/mol at 37°C (Kf = 2.7 × 105) (17). This is stable enough to suppress replicase translation by severalfold. Coat protein binds the hairpin with an affinity of ≈3 × 105 M–1 under physiological conditions and accumulates in cells to 5 to 10 μM (7). Although this binding is fairly weak, the additional stabilization of the hairpin should further repress translation significantly.
The possibility that a bound protein could affect some initiation step other than subunit binding has been considered (12), and two instances have been described in which a repressor protein binds to the 30S-mRNA binary complex and prevents correct binding of (40, 54). In both cases the repressor targets a pseudoknot mRNA structure, with the ribosome binding site located in a single strand linking two pseudoknot helices. Perhaps the protein can stabilize the pseudoknot in a way that affects ribosome access to the initiation codon during the isomerization step (Fig. 1).
In both repression mechanisms, the repressor protein need not bind tightly enough to displace the initiating ribosome. In the first instance, the repressor gets a "boost" from the hairpin secondary structure and only needs to add enough stability to the structure to bring it into the range needed to inhibit translation. In the other mechanism, the protein traps the ribosome on the mRNA and must only prevent some RNA conformational change needed by the ribosome.
RNAs complementary to an mRNA (antisense RNA) are also used for regulation. Expression of the IS10 transposase gene, for example, is inhibited by an antisense RNA complementary to 35 nt covering most of the ribosome binding site (30). Antisense RNA may also repress translation indirectly when bound outside of the initiation site, for instance by shifting the equilibrium between competing secondary structures towards a form that occludes the initiation site, or by promoting mRNA turnover (51).
The intrinsic initiation efficiency of an mRNA is determined by its SD and initiation codon sequences and the secondary and tertiary structures incorporating them. This inherent capacity for protein synthesis may be modulated in vivo by additional factors that are beginning to be quantitatively understood. It has long been known that transcription, translation, and mRNA decay are coupled processes. For instance, a very low level of translational initiation can cause premature termination of transcription (56), and thus lower the concentration of mRNA. The chemical stability of an mRNA also increases with the initiation frequency, as translating ribosomes provide some protection against RNases (47). These effects have recently been quantitated for a set of lacZ genes that have translational initiation signals conferring a 200-fold range of expression (64). The net result of the two coupling mechanisms is that the mRNA level in the cell is proportional to the expression level, and the 200-fold expression range is the result of only a 3-fold range of translational initiation frequency. In a sense, small changes in intrinsic initiation efficiency have been amplified by the coupled effects on mRNA transcription and turnover.
A second factor that may influence the actual in vivo translation rate of a protein is the competition between all the mRNAs in a cell for initiating ribosomes, fMet-tRNA, and initiation factors. Evidence has already been cited that unstructured mRNAs are initiated at the maximum rate possible (11), implying that the translational components are not in limiting supply. But other recent work has shown that induction of a high level of lacZ expression reduces the translation of other genes to varying degrees, presumably because the translational machinery has been monopolized by the lacZ mRNA (62). Thus free ribosomes cannot be in large excess. The experiment provides a novel way of estimating the in vivo strength of a ribosome binding site, since those mRNAs with the largest K 30S values should be least affected by the decrease in free ribosomes.
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