Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2

111

115

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

Nakahigashi

Kubo

Narita

et al. 2002

111

115

“…RF2 overproduced from the wild-type gene is toxic to the cell and shows reduced activity in biochemical assays (17,18), especially with short peptidyl-tRNAs (our observations). RF2ala, which contains a threonine to alanine change at amino acid position 246, as in RF2 from Salmonella typhimurium (19), can be overproduced and yields a protein with enzymatic parameters close to those of RF2 prepared from normal cells: direct comparison with chromosomally produced RF2 shows that k cat is 2-fold lower. This RF2 variant has been used for the accuracy measurements.…”

Section: Methodsmentioning

confidence: 98%

The accuracy of codon recognition by polypeptide release factors

Freistroffer

Kwiatkowski

Buckingham

et al. 2000

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...

“…pSUIQT-RF2* carries the mutant (E167K) RF2 and a tetracycline-resistant marker. A C-terminal histidine tag was marked to RF2 and RF2* by using histidine-tagged PCR primers as described (12,13). Site-directed mutagenesis of RF1 and RF2 was performed by using designed primers coding for the substitutions (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons

Ito

Uno²,

Nakamura³

1998

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG͞UAA and UGA͞UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1͞RF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG͞UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.The termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the ''decoding'' site (A site). Translation termination generally requires two codon-specific polypeptide release factors (RFs), RF1 (for UAG͞UAA) and RF2 (for UGA͞UAA), in prokaryotes (1, 2) and one factor, eRF1 (omnipotent for the three stop codons), in eukaryotes (2-4) (Fig. 1A). However, the mechanism of stop codon recognition by release factors is unknown and represents a long-standing coding problem of considerable interest. It entails protein-RNA recognition rather than the well understood mRNA-tRNA interaction in codon-anticodon pairing (2, 5, 6).The fact that two RFs from prokaryotes exhibit codon specificity suggests that they must interact directly with the codon. On accumulation of RF sequences from different organisms, the conservation of protein motifs has emerged in prokaryotic and eukaryotic RFs, as well as in the C-terminal portion of elongation factor EF-G, a translocase protein that forwards peptidyl tRNA from the A site to the P site on the ribosome (7). The three-dimensional structure of Thermus thermophilus EF-G comprises five subdomains; the C-terminal part, domains III-V, appears to mimic the shapes of the acceptor stem, the anticodon helix, and the T stem of tRNA, respectively (8-10). Furthermore, it appears that an RF region shares homology with domain IV of EF-G, thus constituting a putative ''tRNA-mimicry'' domain necessary for RF binding to the ribosomal A site (7). This mimicry model would explain why RFs recognize stop codons by assuming an anticodonmimicry element in the protein and further suggest that all prokaryotic and eukaryotic RFs evolved from the progenitor of EF-G. RF1 and RF2 are known to be structurally similar, and both read the UAA codon. It might be possible, therefore, to alter mutationally either factor so that its stop codon specificity is altered. In the present study, we mutationally altered RF2 and show that a single amino acid substitution permits it to terminate translation at the UAG stop codon as well as the UGA and UAA ...