tRNA Concentration Research Articles

DoesEscherichia coliOptimize the Economics of the translation Process?

The codon translation rate is usually assumed to be proportional to the cellular concentration of the cognate tRNA, but synonymous codons sharing the same cognate tRNA may be translated at rather different rates. To account for the latter observation, we assume that the translation process is optimized in two respects: (i), the codon demand is optimized with respect to the supply of cognate tRNAs (composition of the tRNA pool); and (ii), for synonymous codon sharing the same cognate tRNA, the usage frequency of each codon correlates optimally with the stability of the codon-anticodon complex. These assumptions allow us to compute the relative rate constants of synonymous codons. Highly expressed genes, which produce 80–90% of the protein mass in theE. colicell, appear to have selected codons which make an optimal use of the tRNA pool. Assuming the optimization criteria were valid, a list of codon translation times (in ms) were derived from available experimental data.

Import of RNA into Leishmania mitochondria occurs through direct interaction with membrane-bound receptors.

Cytoplasmic tRNAs are imported into the kinetoplast mitochondrion of Leishmania, but the mechanism of import is unknown, particularly whether RNA is transferred as a ribonucleoprotein complex through the protein import pathway or by a distinct receptor-mediated mechanism. Using isolated mitochondria, it was shown that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with high affinity to the mitochondrial surface in the absence of soluble protein factors to form an import intermediate. Two classes of binding site of apparent Kd 0.3 and 10 n, respectively, were distinguished. tRNA from Leishmania, but not yeast, competitively inhibited the binding. Northwestern blot analysis revealed the presence of a 15-kDa RNA binding protein on the mitochondrial surface. Whereas receptor binding was resistant to heparin and KCl, internalization was sensitive to both reagents. These results are consistent with the presence of a direct mechanism of receptor-mediated RNA import on Leishmania mitochondria.

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

The existence of a conditional lethal temperature-sensitive mutant affecting peptidyl-tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2- to 3-fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl-tRNA hydrolase activity in the cell at non-permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl-tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.

Control of spoT-dependent ppGpp Synthesis and Degradation in Escherichia coli

Escherichia colihas two ppGpp synthetases, PSI and PSII, encoded by the relAand spoTgenes. The spoT gene also encodes a ppGpp hydrolase. During exponential growth and under various starvation conditions, the level of ppGpp depends on the balance of ppGpp synthetic and degradative activities of spoTgene products. To find out how these two activities respond to different physiological conditions and to learn about the signals involved in these responses, rates of ppGpp synthesis and degradation were determined in an E. coliB/r Δ relAstrain during: (1) multiple amino acid deprivation after a nutritional shift-down from glucose amino acids to glucose minimal medium; (2) carbon source starvation after a “glucose runout”; (3) energy starvation by treatment with sodium azide. To each of these conditions, bacteria responded with a similar gradual accumulation of ppGpp, occurring over a period of 20 to 40 minutes, from the basal level of 4 and 24 pmol/OD 460in glucose amino acids and glucose minimal medium, respectively, to about 100 pmol ppGpp/OD 460unit of culture mass. After multiple amino acid deprivation and during azide treatment, the rate of ppGpp synthesis increased and the rate of ppGpp degradation decreased, but in different proportions by the two kinds of treatment. After glucose runout, both ppGpp synthesis and degradation immediately decreased, but the rate of degradation was reduced more, which caused the accumulation of ppGpp despite its reduced synthesis. ppGpp synthesis required continuous protein synthesis, but ppGpp hydrolysis and its control did not: the rate of ppGpp degradation could be instantly up or down-regulated in response to changes in exogenous amino acid or glucose levels in the absence of protein synthesis. The results suggest that PSII is unstable with an average functional lifetime of 40 seconds or less, and that its activity is generated during or shortly after spoTmRNA translation in response to the availability of amino acids. This regulation is responsible for the growth medium-dependent changes in basal levels of ppGpp. ppGpp hydrolysis is controlled, i.e. inhibited, mainly during conditions of physiological stress, such as multiple amino acid deprivation or energy deprivation. This inhibition can be correlated with an inferred accumulation of uncharged tRNA. Since previous reports have indicated that uncharged tRNA inhibits purified SpoT hydrolase in vitro, it is proposed that ppGpp hydrolase activity is, indeed, controlled by the concentration of uncharged tRNA in the cell. Finally, it was found that neither relC, transcriptional regulation of spoT, nor different translation starts of spoTmRNA are directly involved in the environmental control of SpoT hydrolase and PSII activities.

A 43 kD light-regulated chloroplast RNA-binding protein interacts with the psbA 5? non-translated leader RNA

Expression of the chloroplast psbA gene coding for the D1 protein of Photosystem II is subject to regulation at different levels in higher plants, including control of mRNA accumulation and translation. In dicots, the conserved 5' non-translated leader (5'-UTR) of the psbA mRNA is sufficient to direct the light-dependent translation of the D1 protein. In this report we show that the psbA mRNA 5'-UTR forms a stem-loop structure and binds a 43 kD chloroplast protein (43RNP). Binding of the 43RNP is sensitive to competition with poly(U), but insensitive to high concentrations of tRNA, the RNA homopolymers poly(A), poly(G), poly(C), or poly(A):poly(U) as a double-strand RNA. The 43RNP does not bind efficiently to the psbA mRNA 3' non-translated region, although the RNA sequence is U-rich and folds into a stem-loop. A deletion mutant of the psbA 5'-UTR RNA in which 5' sequences of the stem-loop are removed does not affect 43RNP binding. Together, these properties suggest that the 43RNP binds most effectively to a specific single-strand U-rich sequence preceding the AUG start codon in the psbA mRNA. Binding of the 43RNP is not detectable in plastid protein extracts from 5-day-old dark-grown seedlings, but is detectable in light-grown seedlings as well as mature plants in the light and after shifted to the dark. The 43RNP is therefore a candidate for a regulatory RNA-binding protein that may control the accumulation and/or translation of the psbA mRNA during light-dependent seedling development.

Codon bias in Escherichia coli may modulate translation initiation.

Codons with low corresponding intracellular tRNA concentrations are generally used less frequently (i .e. rare) compared to synonymous codons associated with higher tRNA concentrations 11 1. The differential use of codons with defined tRNA concentrations causes a given mRNA sequence to be translated at variable rates 121. Rare codons with extremely low tRNA concentrations may stall translation until an appropriate tRNA becomes available [31. Translational pauses caused by low tRNA availability may also influence the folding of a nascent polypeptide chain, and it has been proposed that in several proteins (e.g. Rabbit a and p globins, Cytochromes C and Yeast Pyruvate Kinase 131) translational pausing may allow discrete protein domains to fold and interact. Translational pausing of this type therefore complements the kinetics of a protein folding reaction. Translational pauses could also influence the targeting of exported polypeptides. In E . coli proteins are generally exported by interactions of a N-terminal signal sequence with the translocation machinery [4,5,6]. Translational pausing could therefore favour export in two ways. With proteins exported posttranslationally, an increase in time required for translation elongation would also increase the time a nascent polypeptide chain is exposed in the cytoplasm. Chaperone proteins 17) involved specifically in protein translocation (e.g. Sec B [6]) could then bind to the protein chain, increasing both the efficiency of export and the probability of the protein adopting a loose fold suitable for translocation. Proteins exported in a co-translational fashion could also benefit from translational pauses within approximately the first 100 codons. Pauses located around this region would expose the nascent signal sequence in the cytoplasm prior to pausing and this should allow i t to act independently of the un-translated protein. The export process would then be more efficient since direct interactions with the translocation machinery could ensue and the un-translated portion of the protein would not have to be maintained in a translationally competent state. This would be of particular importance in prokaryotic species where there is no evidence for elongation arrest by a Signal Recognition Particle (SRP). We therefore developed analytical software to examine rare codon usage & distribution in a sample containing 47 exported and 46 cytoplasmic polypeptides. The necessary coding regions of Escherichia coli were selected from the Linkage map of E . ccdi K12 181, and subsequently exuacted from the Gembl database using ‘GCG Navigator’ software. To locate potential pause sites we selected 8 rarely used codons 191, with minor concentrations of tRNA [ 11. These were CTA (Leu), ATA (Ile), ACA (Thr), CCT, CCC (Pro), CGG, AGA & AGG (Arg). If translational pausing was a widely used folding strategy, it would be reasonable to assume a longer polypeptide would require more pauses during translation to achieve this. Preliminary data demonstrated that frequency of rare ctdon use appears to be independent of polypeptide chain length. This does not prevent translational pauses regulating protein folding events, but we found no evidence to support this. In order to analyse the role translational pausing may play, detailed structural data on protein domain boundaries is required. Analysis of the distribution of rare The distribution of rare codons in the first 120 codons of 47 exported and 46 cytoplasmic polypeptides.

Nuclear Export Pathways of tRNA and 40 S Ribosomes Include both Common and Specific Intermediates

Different classes of RNAs are exported from Xenopus laevis oocyte nuclei by facilitated pathways. We have performed kinetic competition analyses to investigate the relationship between the export pathways of microinjected tRNA and ribosomal subunits. Saturating concentrations of ribosomal subunits do not compete tRNA export. Thus, the saturable factor in the ribosomal subunit export pathway is not limiting for tRNA export. The co-microinjection of ribosomal subunits did, however, stimulate the rate of tRNA export. Co-injected mRNA also stimulated tRNA export. tRNA export itself displays positive cooperative export kinetics that are abrogated by saturating concentrations of rRNA. These results are consistent with the existence of common high affinity RNA-binding sites that can be titrated with tRNA, rRNA or ribosomal subunits, and mRNA. Furthermore, high concentrations of tRNA are also shown to have moderate inhibitory effects on 40 S subunit export, indicating a lower affinity common intermediate also shared by mRNA.

In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs.

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.

The Escherichia coli fmt gene, encoding methionyl-tRNA(fMet) formyltransferase, escapes metabolic control

The genetic organization near the recently cloned fmt gene, encoding Escherichia coli methionyl-tRNA(fMet) formyltransferase (J. M. Guillon, Y. Mechulam, J. M. Schmitter, S. Blanquet, and G. Fayat, J. Bacteriol. 174:4294-4301, 1992), has been studied. The fmt gene, which starts at a GUG codon, is cotranscribed with another gene, fms, and the transcription start site of this operon has been precisely mapped. Moreover, the nucleotide sequence of a 1,379-bp fragment upstream from fmt reveals two additional open reading frames, in the opposite polarity. In the range of 0.3 to 2 doublings per h, the intracellular methionyl-tRNA(fMet) formyltransferase concentration remains constant, providing, to our knowledge, the first example of a gene component of the protein synthesis apparatus escaping metabolic control. When the gene fusion technique was used for probing, no effect on fmt expression of the concentrations of methionyl-tRNA(fMet) formyltransferase or tRNA(fMet) could be found. The possibility that the fmt gene, the product of which is present in excess to ensure full N acylation of methionyl-tRNA(fMet), could be expressed in a constitutive manner is discussed.

Effects of tRNA(1Leu) overproduction in Escherichia coli.

Strains of Escherichia coli have been produced which express very high levels of the tRNA(1Leu) isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA(1Leu) gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA(1Leu). As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA(1Leu). We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA(1Leu) was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m1G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA(1Leu) operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA(1Leu) levels. This level of tRNA(1Leu) titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.

Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA

Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA. The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured. With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition. The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence. The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage. The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state. Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles.

The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation in Escherichia coli.

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribing lacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of pheA expression by the pheR product in Escherichia coli is mediated through attenuation of transcription.

In Escherichia coli, the expression of the phenylalanine biosynthetic enzyme chorismate mutase/prephenate dehydratase, encoded by pheA, is elevated in strains carrying pheR mutants. By constructing a series of pheA''cat'lacZ fusions with different endpoints for deletions of the pheA regulatory DNA, the site of action of the pheR product on pheA expression was determined to be the pheA attenuator. Southern blot analysis of chromosomal DNA from a pheR374 strain showed it to carry a deletion of pheR and the flanking DNA on each side. This deletion resulted in a decrease of approximately 30% in the intracellular concentration of tRNA(Phe), the pheR product. The expression of the pheST operon, which encodes the two subunits of phenylalanyl-tRNA synthetase and which is also regulated by attenuation control involving phenylalanyl-tRNA(Phe), was increased 5-fold by the pheR374 allele. No effect of pheR on pheST expression was seen in a pheST(att-) strain. It was concluded that the elevated expression of pheA and pheST in pheR mutants is a consequence of a lower frequency of transcription termination in the attenuator caused by lower levels of phenylalanyl-tRNA(Phe).

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules.

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.

Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins

Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).

Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.

Time of action of 4·5 S RNA in Escherichia coli translation

A new class of suppressor mutants helps to define the role of 4·5 S RNA in translation. The suppressors reduce the requirement for 4·5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4·5 S RNA enters translation. The release of 4sd5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4·5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4·5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4·5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4·5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.

Rates of aminoacyl-tRNA selection at 29 sense codons in vivo

We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate. By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli. For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis. Therefore, the frameshift assay probably measures this process in vivo. Observed rates for aminoacyl-tRNA selection span a 25-fold range. Therefore, the time required to transit different codons in vivo probably differs substantially. Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons. This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons. However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection. Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons. Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly. Other factors are therefore more important than speed for some codons. Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes. Instead, there is a slight preference for slower aminoacyl-tRNA selection. The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex. Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing. However, no more general relationship is evident between codon/ anticodon composition and rate of aminoacyl-tRNA selection. The frameshift method can be extended to all codons.

On the non‐liner Eadie plots of the tRNA kinetics and non‐liner Dixon plots of the PPi inhibition kinetics of the aminoacyl‐tRNA synthetases

A model of the aminoacyl-tRNA synthetase reaction was analyzed by deriving a rate equation, and by calculating the aminoacylation rates at various values of the rate and equilibrium constants. The model specially contained the possibilities that (1) the activation of the amino acid occurs either with bound or non-bound tRNA, and that (2) the transfer of the aminoacyl moiety from the aminoacyl adenylate to tRNA occurs either with bound or non-bound PPi. The analysis showed that the Eadie plots (tRNA as the variable substrate) are straight lines only if the rates of the activation reactions with bound and non-bound tRNA are equal. Otherwise the Eadie plots can be either curved upwards or downwards. The Dixon plots of the PPi inhibition are straight lines only if PPi must be dissociated from the enzyme before the transfer reaction. The conditions under which the Kiapp values are much lower than the dissociation constants for PPi are met if the transfer reaction is relatively slow and the reverse reaction of the activation (pyrophosphorolysis) is fast, and if the tRNA concentration is low.