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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.
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PMID:Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. 1044 76

The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.
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PMID:A tripeptide 'anticodon' deciphers stop codons in messenger RNA. 1068 8

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.
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PMID: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. 1183 Jun 50

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.
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PMID:Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes. 1190 89

E. coli mutants of RF1 and RF2, in which the universal GGQ motif is changed to GAQ, are slow in peptide release from ribosomes. Other kinetic properties are unchanged, suggesting that the GGQ motif is in contact with the peptidyl-transferase center. Deacylated tRNA terminates protein synthesis codon specifically, indicating that the CCA end of tRNA and the GGQ motif operate similarly. Addition of a mutant factor to a pretermination ribosomal complex stimulates exchange of RF3-bound GDP with free GDP, but binding of GTP to RF3 and GTP hydrolysis requires peptide chain release. Therefore, the sequence of steps during termination of translation is regulated by removal of the polypeptide, an event that might trigger a conformational change in the ribosome.
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PMID:Release of peptide promoted by the GGQ motif of class 1 release factors regulates the GTPase activity of RF3. 1241 23

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.
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PMID:Structure of the Escherichia coli ribosomal termination complex with release factor 2. 1255 80

Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain release factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.
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PMID:Termination of translation: interplay of mRNA, rRNAs and release factors? 1251 23

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyltransferases (MTases). In the present study, we sequenced a 5026 bp DNA fragment just downstream of the PgPepO gene reported previously. The DNA sequence analysis revealed three ORFs. The ORF2 gene encoded a protein of 294 amino acids with a calculated molecular weight of 32,160 Da. The deduced amino acid sequence of the ORF2 gene exhibited a significant similarity to sequence of HemK from E. coli (35% identical residues). The ORF2 gene complemented an E. coli hemK mutant. Thus, ORF2 was named PgHemK. From the point of veiw of our recent finding, that E. coli HemK catalyses the methylation of polypeptide chain release factors such as RF1 and RF2, we postulated that PgHemK might function as a protein MTase containing the DNA MTase motif.
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PMID:Cloning and sequencing a HemK-family gene in Porphyromonas gingivalis. 1275 33

Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.
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PMID:Visualization of release factor 3 on the ribosome during termination of protein synthesis. 1498 67

Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N(5)-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N(5)-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1.eRF3.GTP.
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PMID:The glutamine residue of the conserved GGQ motif in Saccharomyces cerevisiae release factor eRF1 is methylated by the product of the YDR140w gene. 1550 72


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