Anticodon Switching Changes the Identity of Methionine and Valine Transfer RNAs

147

150

The precision with which individual termination codons in mRNA are recognized by protein release factors (RFs) has been measured and compared with the decoding of sense codons by tRNA. An Escherichia coli system for protein synthesis in vitro with purified components was used to study the accuracy of termination by RF1 and RF2 in the presence or absence of RF3. The efficiency of factor-dependent termination at all sense codons differing from any of the three stop codons by a single mutation was measured and compared with the efficiency of termination at the three stop codons. RF1 and RF2 discriminate against sense codons related to stop codons by between 3 and more than 6 orders of magnitude. This high level of accuracy is obtained without energy-driven error correction (proofreading), in contrast to codon-dependent aminoacyl-tRNA recognition by ribosomes. Two codons, UAU and UGG, stand out as hotspots for RF-dependent premature termination.protein synthesis ͉ translational processivity ͉ premature termination S ixty-one of the 64 base triplets in the genetic code are sense codons, which are translated in bacteria to the 20 standard amino acids in proteins by about 50 aminoacyl-tRNAs. The remaining three codons usually signal termination of translation and the release of completed proteins from ribosomes by release factors RF1 and RF2. Efficient translation requires both rapid binding to their respective codons of cognate aminoacyl-tRNAs, in ternary complex with elongation factor EF-Tu and GTP, and fast termination of protein synthesis at stop signals by RFs. In addition to fast signal processing, codon translation must attain a high level of accuracy so that nonacceptable amino acid replacements in nascent proteins do not impair cell growth and viability. Similarly, misrecognition by RFs of sense codons as stop signals leads to truncated proteins, an energetically costly event, the frequency of which also must be contained.Pauling was among the first to reflect on the errors that must occur during protein synthesis because of the limited maximum differences in interaction standard free energies between different pairs of amino acids, and he concluded that amino acid substitutions theoretically should occur at frequencies in the percent range (1). Though later shown to be overly pessimistic (2), these predictions led to theoretical studies showing that enzymatic selection can, under certain conditions, be more accurate than the instrinsic selectivity of a single step.The accuracy (A) of simple enzyme selection schemes, defined as the probability of correct product formation divided by the probability of an error at equal concentrations of cognate and noncognate substrates, is limited by the standard free energy difference between the transition state leading to product formation and ground state for cognate (⌬G c ) and noncognate (⌬G nc ) substrates by the inequality. The precision of codon translation depends on differences in H-bond energies between cognate and noncognate tRNAcodon interactions, though other i...

Section: Resultsmentioning

confidence: 99%

The accuracy of codon recognition by polypeptide release factors

Freistroffer

Kwiatkowski

Buckingham

et al. 2000

147

150

“…Secondly, the high K M for amino acids for many synthetases makes it very difficult to perform reactions at a saturating concentration of amino acids without the use of prohibitive amounts of radioactivity. Because both the amino acid and tRNA often participate in the organization of the active sites of aaRS (3,(28)(29)(30)(31)(32), a saturating concentration of one of the substrates is required for the proper determination of the kinetic constants for the other substrate. Finally, the sensitivity of the traditional assay is limited, especially with poor tRNA substrates that show low rates of aminoacylation.…”

Section: Resultsmentioning

confidence: 99%

“…The second involves assaying aminoacylation activity of mutant tRNAs made by transcription in vitro (2). Both methods reveal a limited number of nucleotides critical for aminoacylation, the importance of which was confirmed by performing ''swap'' experiments in which the proposed nucleotides were transplanted into the body of another tRNA (2,3). Subsequent analysis of aminoacylation rate in vitro or sequencing of the reporter proteins in vivo was used to confirm that change in tRNA identity had occurred.…”

mentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

122

163

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3 -32 P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA Ala . The effect of tRNA Ala identity mutations on both aminoacylation efficiency (kcat͞KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat͞KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA Ala identity.T wo major approaches have been developed to identify nucleotides required for tRNA recognition by aminoacyl-tRNA (aa-tRNA) synthetases (aaRSs). The first involves introducing a suppressor anticodon into a tRNA of interest and then examining the suppressor activity of the point mutations in vivo (1). The second involves assaying aminoacylation activity of mutant tRNAs made by transcription in vitro (2). Both methods reveal a limited number of nucleotides critical for aminoacylation, the importance of which was confirmed by performing ''swap'' experiments in which the proposed nucleotides were transplanted into the body of another tRNA (2, 3). Subsequent analysis of aminoacylation rate in vitro or sequencing of the reporter proteins in vivo was used to confirm that change in tRNA identity had occurred. These two methods generally agree reasonably well with each other and correlate well with observed base-specific contacts in cocrystal structures of tRNA-aaRS complexes (4-6). However, there are several examples where the conclusions derived from in vitro and in vivo approaches disagree, the most notable of which is the recognition of tRNA Ala by Escherichia coli alanyl-tRNA synthetase (AlaRS). Although no cocrystal structure is available, in vitro experiments with truncated RNA substrates (7, 8) and mutant tRNA Ala (9) as well as identity-swap experiments (10, 11) strongly suggested that the G3⅐U70 pair is the major identity element of tRNA Ala . The deleterious effect of mutations of this base pair significantly exceeds those of other tRNA Ala recognition elements (8, 9, 12-15) and clearly was indicative of the predominant role of this base pair and in particular the exocyclic amino group of G3 in recognition. In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discre...

“…Each tRNA was mutated to contain the tRNA Val anticodon, G34A35C36, and the C38A mutation was introduced into tRNA Asp and tRNA Glu . These mutations introduce important identity determinants expected to improve misacylation by E. coli ValRS and yeast PheRS (6)(7)(8)(9)(10)18). Because EF-Tu binds to the acceptor stem and the T arm of tRNA (19,20), these anticodon modifications are not expected to affect binding.…”

Section: Methodsmentioning

confidence: 99%

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

112

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)⅐GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from ؊11.7 kcal͞mol for Val-tRNA Glu to ؊8.1 kcal͞mol for Val-tRNA Tyr , clearly establishing EF-Tu⅐GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of koff varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism. E longation factor Tu (EF-Tu) binds GTP and aminoacyl tRNA (aa-tRNA) to form a ternary complex that subsequently binds ribosome and participates in codon-directed binding of the aa-tRNA to the ribosomal A site. Although EFTu⅐GTP binds poorly to tRNAs lacking the esterified amino acid (1), the protein is generally considered to lack specificity because it binds all elongator aa-tRNAs with a similar affinity (2-4). However, recent experiments have shown that EF-Tu⅐GTP exhibits substantial specificity for both the esterified amino acid and the tRNA body of aa-tRNAs (5). This specificity was previously unappreciated because the contributions of the amino acid and the tRNA body to the overall affinity are arranged in a compensatory manner such that the cognate aa-tRNAs all bind with similar affinities. However, misacylated tRNAs were found to bind EF-Tu⅐GTP with a wide range of affinities that were either tighter or weaker than the cognate aa-tRNAs (5). The four different tRNA bodies that were tested displayed about a 100-fold range of K D values with Thermus thermophilus EFTu⅐GTP when each was esterified with the same amino acid. The same range of K D values was observed when the four tRNAs were esterified with a different common amino acid, clearly establishing that EF-Tu shows specificity toward the tRNA body. However, the four tRNAs used in these experiments (Escherichia coli tRNA Ala , tRNA Gln , tRNA Val , and yeast tRNA Phe ) have very similar overall architectures, raising the possibility that EFTu⅐GTP could exhibit a much larger range of affinities with tRNAs of different architectures.Experiments presented here take advantage of the important role of the anticodon as a identity element for valyl-tRNA synthetase (ValRS) and phenylalanyl-tRNA synthetase (PheRS) (6-10) to prepare 21 different E. coli tRNAs that were misacylated with either valine or phenylalanine. By comparing the affinities of the different tRNAs esterified with a common amino acid, the specificity of EF-Tu⅐GTP for the different tRNA bodies was established. Materials and MethodsE. coli...