Release Factors eRF1 and RF2

Ma, Buyong; Nussinov, Ruth

doi:10.1074/jbc.m407412200

Cited by 40 publications

(18 citation statements)

References 34 publications

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“…Molecular modeling showed that eRF1 can undergo a significant conformational change in the ribosome (37,38), as suggested earlier (36). Because of this change, the NIKS and YxCxxxF motifs become proximal to the stop codon at the A site (38), which implies that, for these eRF1s, a probable mechanism for stop codon restriction is associated with an interaction in space between discriminator and recognition sites, leading to local conformational changes of the latter.…”

Section: Discussion Functional Assays For Measuring Decoding Specificmentioning

confidence: 91%

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors

Lekomtsev

Kolosov

Bidou

et al. 2007

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

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In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universalcode ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124 -131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons. ciliated protozoa ͉ dual gene reporter system ͉ eukaryote class-1 polypeptide release factors ͉ interdomain and intradomain protein chimeras ͉ stop codon decoding I n the universal genetic code, three stop codons (UAA, UAG, and UGA) located at the termini of mRNA sequences are decoded at the termination step of translation by class-1 polypeptide release factors (RF) (reviewed in refs. 1-3). However, in organisms with variations in the genetic code, like ciliates, class-1 factors are able to decode only one or two stop codons with the remaining stop codon(s) reassigned to encode certain amino acids (for review, see refs. 4 and 5). The molecular mechanisms that restrict stop codon recognition are entirely unknown and represent major unresolved problems in molecular biology and genetics. In eukaryotes with the standard code, a single class-1 RF, designated eukaryote class-1 polypeptide release factor (eRF1), decodes all three stop codons. Stop codon decoding results in signal transduction from the small to the large ribosomal subunit leading to cleavage of peptidyl-tRNA at the peptidyl transferase center of the ribosome.The eRF1 protein family is highly conserved and similar to archaeal class-1 RFs but differs profoundly from bacterial class-1 RFs (6-8). The only known structural element common to all class-1 RFs is a univer...

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Section: Discussion Functional Assays For Measuring Decoding Specificmentioning

confidence: 91%

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors

Lekomtsev

Kolosov

Bidou

et al. 2007

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

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“…Cryo-EM and recent X-ray structure analysis of Escherichia coli and Thermus thermophilus ribosomes in complex with the corresponding class I RFs resolved this conflict by identification of elongated RFs bound to terminating ribosomes (22,26,27). This model of conformational adaptation of RFs upon ribosomal binding is supported by a molecular dynamic study (28). …”

Section: Introductionmentioning

confidence: 78%

“…Opening of the compact core exposes conserved hydrophobic residues at the interface between domains III and II·IV and increases the water accessible surface by 15% (27). It was suggested that the conformational transition of RF2 between free and ribosome-bound states are controlled by protonation of conserved histidines (28). Indeed, it can be expected that the overall negative charge of the ribosome will enhance the histidine protonation of RF2.…”

Section: Discussionmentioning

confidence: 99%

Release Factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Redecke

Svergun

Konarev

et al. 2007

Nucleic Acids Research

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Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 Å distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II·III·IV is required, which contrasts the known compact RF crystal structures. The crystal structure of Thermus thermophilus RF2 was determined and compared with solution structure of T. thermophilus and Escherichia coli RF2 by microcalorimetry, circular dichroism spectroscopy and small angle X-ray scattering. The structure of T. thermophilus RF2 in solution at 20°C is predominantly compact like the crystal structure. Thermodynamic analysis point to an initial melting of domain I, which is independent from the melting of the core. The core domains II·III·IV melt cooperatively at the respective physiological temperatures for T. thermophilus and E. coli. Thermodynamic analyses and the X-ray scattering results for T. thermophilus RF2 in solution suggest that the compact conformation of RF2 resembles a physiological state in absence of the ribosome.

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“…Indeed, the NIKS and GGQ domains are separated by 100 Å in the crystal structure, whereas the distance between the codon in the A-site and the peptidyl center is around 73 Å. A conformational change has been modeled to create a closed form, which fits the range needed for functional binding of eRF1 within the ribosome (33). The closed conformation of eRF1 involves motion of domain 2 relative to other domains.…”

Section: Discussionmentioning

confidence: 99%

Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

Hatin¹,

Fabret²,

Rousset³

et al. 2009

Nucleic Acids Research

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Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3—a GTPase protein—probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

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Release Factors eRF1 and RF2

Cited by 40 publications

References 34 publications

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors

Release Factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies

Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

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