scholarly journals Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

2018 ◽  
Vol 115 (3) ◽  
pp. E382-E389 ◽  
Author(s):  
Thomas Philipp Hoernes ◽  
Nina Clementi ◽  
Michael Andreas Juen ◽  
Xinying Shi ◽  
Klaus Faserl ◽  
...  
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Stop Codons ◽  
Codon Recognition ◽  
Chemically Modified ◽  
Peptide Release ◽  
Stop Codon Recognition ◽  
A Site ◽  
Chemical Groups ◽  
Insight Into

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

scholarly journals Conformational control of translation termination on the 70S ribosome

2017 ◽  
Author(s):  
Egor Svidritskiy ◽  
Andrei A. Korostelev
Keyword(s):  
Conformational Changes ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Peptide Release ◽  
Stop Codon Recognition ◽  
70S Ribosome ◽  
A Site

AbstractTranslation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate translation termination. If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codon-recognition domain is fully engaged in stop-codon recognition in the decoding center. RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.

scholarly journals Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (8) ◽  
pp. 1378-1387 ◽  
Author(s):  
Adam K. Kallmeyer ◽  
Kim M. Keeling ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Number Of Factors ◽  
Stop Codon Recognition

ABSTRACT Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

scholarly journals PABP enhances release factor recruitment and stop codon recognition during translation termination

2016 ◽  
Vol 44 (16) ◽  
pp. 7766-7776 ◽  
Author(s):  
Alexandr Ivanov ◽  
Tatyana Mikhailova ◽  
Boris Eliseev ◽  
Lahari Yeramala ◽  
Elizaveta Sokolova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Stop Codon Recognition

scholarly journals Two-step model of stop codon recognition by eukaryotic release factor eRF1

2013 ◽  
Vol 41 (8) ◽  
pp. 4573-4586 ◽  
Author(s):  
Polina Kryuchkova ◽  
Alexander Grishin ◽  
Boris Eliseev ◽  
Anna Karyagina ◽  
Ludmila Frolova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Recognition ◽  
Step Model ◽  
Stop Codon Recognition

scholarly journals Kinetics of Stop Codon Recognition by Release Factor 1

Biochemistry ◽  
2009 ◽  
Vol 48 (47) ◽  
pp. 11178-11184 ◽  
Author(s):  
Byron Hetrick ◽  
Kristin Lee ◽  
Simpson Joseph
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Recognition ◽  
Stop Codon Recognition ◽  
Kinetics Of

Functional characterization of polypeptide release factor 1b in the ciliate Euplotes

2010 ◽  
Vol 30 (6) ◽  
pp. 425-431 ◽  
Author(s):  
Yan Wang ◽  
Baofeng Chai ◽  
Wei Wang ◽  
Aihua Liang
Keyword(s):  
Stop Codon ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Class I ◽  
Release Factor ◽  
Amino Acid Motif ◽  
Codon Recognition ◽  
Stop Codon Recognition ◽  
Higher Eukaryotes

In higher eukaryotes, RF-I (class I release factor) [eRF1 (eukaryotic release factor 1)] is responsible for stop codon recognition and promotes nascent polypeptide release from the ribosome. Interestingly, two class I RFs, eRF1a and eRF1b, have been identified among the ciliates Euplotes, which are variant code organisms. In the present study, we analysed the comparative expression of eRF1a and eRF1b in Euplotes cells, demonstrating that the expression of eRF1b was higher than that of eRF1a. An interaction between eRF1b and eRF3 was confirmed, suggesting that an eRF1b function is facilitated by eRF3. Co-localization of both eRF1s indicated that they function in the same subcellular location in Euplotes cells. We also analysed the characteristics of stop codon discrimination by eRF1b. Like eRF1a, eRF1b recognized UAA and UAG as stop codons, but not UGA. This finding disagreed with the deduced characteristics of eRF1a/eRF1b from the classic hypothesis of ‘anticodon-mimicry’ proposed by Muramatsu et al. [Muramatsu, Heckmann, Kitanaka and Kuchino (2001) FEBS Lett. 488, 105–109]. Mutagenesis experiments indicated that the absolutely conserved amino acid motif ‘G31T32’ (numbered as for human eRF1) in eRF1b was the key to efficient stop codon recognition by eRF1b. In conclusion, these findings support and improve the ‘cavity model’ of stop codon discrimination by eRF1 proposed by Bertram et al. [Bertram, Bell, Ritchie, Fullerton and Stansfield (2000) RNA 6, 1236–1247] and Inagaki et al. [Inagaki, Blouin, Doolittle and Roger (2002) Nucleic Acids Res. 30, 532–544].

Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor

2013 ◽  
Vol 91 (3) ◽  
pp. 155-164 ◽  
Author(s):  
Lijun Xu ◽  
Yanrong Hao ◽  
Cui Li ◽  
Quan Shen ◽  
Baofeng Chai ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Reassignment ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Class 1 ◽  
Stop Codon Recognition ◽  
Stop Codon Reassignment

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

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