Quantitative analysis of ribosome–mRNA complexes at different translation stages

Shirokikh, Nikolay E.; Alkalaeva, Elena; Vassilenko, Konstantin S.; Afonina, Zhanna A.; Alekhina, Olga M.; Kisselev, Lev L.; Spirin, Alexander S.

doi:10.1093/nar/gkp1025

Cited by 36 publications

(54 citation statements)

References 28 publications

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“…Compaction of mRNA in the A site probably results in pulling downstream mRNA further into the mRNA channel. This is consistent with protection of two additional nucleotides of 3’ mRNA upon eRF1 binding to the ribosome 16,17 .…”

supporting

confidence: 85%

Structural basis for stop codon recognition in eukaryotes

Brown

Shao

Murray

et al. 2015

Nature

259

392

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Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UGA, UAA, or UAG. Release factors recognise stop codons in the ribosomal A site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases1. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognise all three stop codons2. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here, we present electron cryo-microscopy (cryo-EM) structures at 3.5 – 3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A site. Binding of eRF1 flips nucleotide A1825 of 18S rRNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A site, where it is stabilised by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during tRNA selection to drive mRNA compaction. Stop codons are favoured in this compacted mRNA conformation by a hydrogen-bonding network with essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination3,4.

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supporting

confidence: 85%

Structural basis for stop codon recognition in eukaryotes

Brown

Shao

Murray

et al. 2015

Nature

259

392

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“…We observed toeprints +17 to +19 nt downstream of AUG ( Figure 10C) which is a hallmark of ribosome recruitment to the AUG and stable ribosome-RNA complex formation. Depending on the distribution of fluorescence intensity (+17< +18 > +19) (Shirokikh et al, 2010) we further confirmed that indeed 80S initiation complex was isolated which was formed on XIAP IRES RNA in eIF2 phosphorylation condition ( Figure 10C). The initiation complex can be further purified by sucrose density gradient separation.…”

Section: Formation and Isolation Of Xiap Ires Initiation Complex By Rsupporting

confidence: 72%

RNA Affinity Chromatography

Thakor¹,

Holčı́k²

2012

Affinity Chromatography

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“…The assembly efficiency was determined by primer extension inhibition (toeprinting) using fluorescently labeled DNA primers and capillary electrophoresis (Figure 1D) (19,20). …”

Section: Resultsmentioning

confidence: 99%

Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits

Ivanova¹,

Berger²,

Scherrer³

et al. 2015

Nucleic Acids Research

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The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mRNAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection.

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Quantitative analysis of ribosome–mRNA complexes at different translation stages

Cited by 36 publications

References 28 publications

Structural basis for stop codon recognition in eukaryotes

Structural basis for stop codon recognition in eukaryotes

RNA Affinity Chromatography

Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits

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