Binding of misacylated tRNAs to the ribosomal A site

To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aa-tRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodon.codon interaction was maintained, thus removing concern that different anticodon.codon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aa-tRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.     

Contribution of the esterified amino acid to the binding of aminoacylated tRNAs to the ribosomal P- and A-sites

Crystallographic studies suggest that the esterified amino acid of aminoacyl tRNA make contacts with the ribosomal A-site but not in the P-site. Biochemical evidence indicating a thermodynamic contribution of the esterified amino acid to binding aminoacyl-tRNA to either the ribosomal P- and A-sites has been inconsistent, partly because of the labile nature of the aminoacyl linkage and the long times required to reach equilibrium. Measuring the association and dissociation rates of deacylated and aminoacylated tRNAs to the A-site and P-site of E. coli ribosomes afforded an accurate estimate of the contribution of the amino acid. While esterified phenylalanine or methionine has no effect on the affinity of tRNA to the P-site, an esterified pheylalanine stabilizes binding to the A-site by 7 kJ/mol, in agreement with the contacts observed in the X-ray crystal structure. In addition, it was shown that the presence of an esterified amino acid in one ribosomal site does not affect the binding of an aa-tRNA to the other site.     

Is the sequence-specific binding of aminoacyl-tRNAs by EF-Tu universal among bacteria?

Three base pairs in the T-stem are primarily responsible for the sequence-specific interaction of tRNA with Escherichia coli and Thermus thermophilus EF-Tu. While the amino acids on the surface of EF-Tu that contact aminoacyl-tRNA (aa-tRNA) are highly conserved among bacteria, the T-stem sequences of individual tRNA are variable, making it unclear whether or not this protein-nucleic acid interaction is also sequence specific in other bacteria. We propose and validate a thermodynamic model that predicts the ΔG° of any tRNA to EF-Tu using the sequence of its three T-stem base pairs. Despite dramatic differences in T-stem sequences, the predicted ΔG° values for the majority of tRNA classes are similar in all bacteria and closely match the ΔG° values determined for E. coli tRNAs. Each individual tRNA class has evolved to have a characteristic ΔG° value to EF-Tu, but different T-stem sequences are used to achieve this ΔG° value in different bacteria. Thus, the compensatory relationship between the affinity of the tRNA body and the affinity of the esterified amino acid is universal among bacteria. Additionally, we predict and validate a small number of aa-tRNAs that bind more weakly to EF-Tu than expected and thus are candidates for acting as activated amino acid donors in processes outside of translation.     

Amino acid specificity in translation

Recent structural and biochemical experiments indicate that bacterial elongation factor Tu and the ribosomal A-site show specificity for both the amino acid and the tRNA portions of their aminoacyl-tRNA (aa-tRNA) substrates. These data are inconsistent with the traditional view that tRNAs are generic adaptors in translation. We hypothesize that each tRNA sequence has co-evolved with its cognate amino acid, such that all aa-tRNAs are translated uniformly.     

Specificity of the ribosomal A site for aminoacyl-tRNAs

Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2′-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k_off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.     

3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-32P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3'-32P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.     

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins. Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA.     

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.     

Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

Conserved features of coordinately regulated E. coli promoters

E. coli promoters which are coordinately regulated in response to amino acid limitation contain conserved nucleotide sequences immediately 3' to -10 region. These sequences contain predominantly either GC or AT residues depending on whether the response is respectively negative or positive. Certain classes of promoters also contain conserved sequences upstream of the primary promoter. In tRNA genes these sequences could act as a secondary polymerase binding site.     

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.     

The second to last amino acid in the nascent peptide as a codon context determinant.

Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon. The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli. A 30-fold difference in readthrough of the UGA codon was observed. Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction. Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant. This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing. The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNA(Trp) as compared with a suppressor form of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.     

Alteration of the intracellular concentration of aminoacyl-tRNA synthetases and isoaccepting tRNAs during amino-acid limited growth in Escherichia coli.

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a depression of the threonyl-tRNA synthetase and isoleucyl-tRNA synthetase respectively. During this amino-acid-limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed-phase-5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl-tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs. So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibiotics. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation. Thus the observed changes in the profiles of isoaccepting tRNAs during amino acid limitation signal a meaningful biological function of those newly or increasingly occurring isoaccepting tRNAs. During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine-limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.     

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.     

Requirement for C-terminal Extension to the RNA Binding Domain for Efficient RNA Binding by Ribosomal Protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

Predicting the binding affinities of misacylated tRNAs for Thermus thermophilus EF-Tu.GTP

The free energies for the binding of 20 different unmodified Escherichia coli elongator aminoacyl-tRNAs to Thermus thermophilus elongation factor Tu (EF-Tu) were determined. When combined with the binding free energies for the same tRNA bodies misacylated with either valine or phenylalanine determined previously [Asahara, H., and Uhlenbeck, O. C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3499-3504], these data permit the calculation of the contribution of each esterified amino acid to the total free energy of binding of the complex. The two data sets can also be used to calculate the free energy of binding of EF-Tu to any misacylated E. coli tRNA, and the values agree well with previously published experimental values. In addition, a survey of active misacylated suppressor tRNAs suggests that a minimal threshold of binding free energy for EF-Tu is required for suppression to occur.     

Aminoacylation of tRNAs as critical step of protein biosynthesis.

Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.     

Topography of the E site on the Escherichia coli ribosome.

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.