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

Mottagui‐Tabar, Salim; Björnsson, Ásgeir; Isaksson, Leif A.

doi:10.1002/j.1460-2075.1994.tb06255.x

Cited by 122 publications

(147 citation statements)

References 40 publications

(35 reference statements)

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“…Evidence for in vivo termination at UAG also was demonstrated by using an assay based on a gene that codes for three identical engineered antibody binding B domains of protein A from Staphylococcus aureus (15,16). The sequence with the UAG codon was inserted into a linker between the segments coding for the second and third identical IgG binding domains (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In fact, RF2 genes of prokaryotes are associated with autogenous ϩ1 frameshift control at the internal UGA signal (26). If this mechanism is essential to control RF2 distinctly from RF1, it (15,16). (C) Influence on UAG readthrough of expression of various RF constructs.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of Protein Products of the 3A Gene. E. coli test strains were transformed with the 3AЈ reporter plasmid pAB96 (15,16). Transformants were grown in LB media containing selective antibiotics and IPTG (1 mM), and exponentially growing cells were examined for the synthesis of 3AЈ and 2AЈ proteins as described (13).…”

Section: Methodsmentioning

confidence: 99%

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

Ito

Uno²,

Nakamura³

1998

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Ito

Uno²,

Nakamura³

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…The 5h nucleotide context of a stop codon also influences how efficiently a stop codon directs termination. In E. coli, this operates partly through the identity of the penultimate amino acid residue in the nascent peptide, which influences termination efficiency by up to 30-fold (Mottagui-Tabar et al, 1994). Efficient termination events are associated with penultimate amino acid residues that are basic in nature.…”

Section: The Termination Signal : Stop Codons and The Role Of Flankinmentioning

confidence: 99%

Endless possibilities: translation termination and stop codon recognition

et al. 2001

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OverviewThe process of protein synthesis can be divided into three main phases : initiation, during which the ribosomal subunits join the mRNA and locate the AUG initiator codon ; elongation, during which sense codons are decoded and the bulk of the polypeptide is made ; and termination, during which a stop codon directs the release of the completed polypeptide from the ribosome. Whereas the general principles of sense codon decoding by transfer RNAs are well established, a clear picture of translation termination and stop codon recognition has hitherto been lacking. Recently however, the use of optimized, complex, reconstituted in vitro termination reactions to identify the roles of key termination factors, and the solution of tertiary structures of termination factors, has allowed a reappraisal of termination factor structure and function. This review will describe these recent advances, many of which have resulted from studies of termination in micro-organisms, and in the light of the new information, discuss models for the mechanism of termination and recognition of the stop codon. While the mechanism of peptide chain termination is normally effective, stop codons are not always recognized efficiently, and during the translation of certain viral RNAs can be suppressed or read through, resulting in the expression of additional coding information. How stop codons are reassigned to encode ' sense ' at a given frequency in some RNAs will be reviewed in the context of current understanding of termination and stop codon recognition mechanisms. The translation termination apparatus in eukaryotes and prokaryotesDuring translation termination, a stop codon located in the ribosomal A-site is recognized by a release factor or release factor complex, which binds the ribosome and triggers release of the nascent peptide. In eukaryotes, translation is terminated by a heterodimer consisting of two proteins, release factors eRF1 and eRF3, which interact in vivo (Frolova et al., 1994 ;Zhouravleva et al., 1995 ;Stansfield et al., 1995). eRF1 recognizes all three stop codons and triggers peptidyl-tRNA hydrolysis by the ribosome, releasing the nascent peptide (Frolova et al., 1994 ; Drugeon et al., 1997). Eukaryote termination efficiency is enhanced by the GTPase release factor eRF3, the second component of the heterodimer eRF complex. In response to a stop codon in the ribosomal A-site, formation of a quaternary complex comprising the ribosome, eRF1, GTP and eRF3 triggers GTP hydrolysis and enhances the rate of peptidyl release (Zhouravleva et al., 1995 ; Frolova et al., 1994 ; Fig. 1). In yeast, eRF1 and eRF3 are encoded by essential genes (SUP35 and SUP45, respectively), mutations in which produce nonsense suppression phenotypes (Stansfield & Tuite, 1994).In contrast to eukaryotes, the role of stop codon recognition during translation termination in eubacteria is divided between two so-called class 1 release factors, RF1 and RF2, which in Escherichia coli are encoded by the essential prfA and prfB genes, respectively (Scolni...

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“…The orientation of the factor to the stop signal at this key invariant position was clearly affected by the adjoining tRNA, specified by the codon in the upstream part of the defined sequence element. This can explain why the last codon (the NNN adjacent to the stop codon in the sequence element) was highly influential on the efficiency of stop codon readthrough as determined in a series of studies by Isaksson and colleagues [44,45]. Moreover, if a Shine and Dalgarno element that can base pair with the 16S rRNA is placed upstream of the stop codon, as is found in the frameshift site for the prfB gene encoding RF2, then the orientation of the first base of the stop codon to the decoding RF is again affected as determined from the site-directed crosslinks ( Figure 6B).…”

Section: Is the Decoding Rf More Promiscuous Than A Trna In Its Contamentioning

confidence: 99%

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

et al. 2007

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The decoding release factor (RF) triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre (PTC) of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure: the anticodon loop and the loop at the tip of helix a5. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.

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

Cited by 122 publications

References 40 publications

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

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

Endless possibilities: translation termination and stop codon recognition

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

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