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The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.ORCID iD: 0000-0003-0742-3906
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA.
Department of Biological Sciences, Barnard College, New York, USA.
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 2579Article in journal (Refereed) Published
Abstract [en]

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Place, publisher, year, edition, pages
2019. Vol. 10, article id 2579
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-382011DOI: 10.1038/s41467-019-10608-zISI: 000471224100009PubMedID: 31189921OAI: oai:DiVA.org:uu-382011DiVA, id: diva2:1306993
Funder
NIH (National Institute of Health), R01 GM55440NIH (National Institute of Health), R01 GM29169Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

De 3 första författarna delar förstaförfattarskapet.

Title in thesis list of papers: The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

Available from: 2019-04-25 Created: 2019-04-25 Last updated: 2019-07-05Bibliographically approved
In thesis
1. Accuracy of protein synthesis and its tuning by mRNA modifications
Open this publication in new window or tab >>Accuracy of protein synthesis and its tuning by mRNA modifications
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ribosome is a large macromolecular complex that synthesizes all proteins in the cell in all kingdoms of life. Proteins perform many vital functions, ranging from catalysis of biochemical reactions to muscle movement. It is essential for cells and organisms that proteins are synthesized rapidly and accurately.

This thesis addresses two questions regarding the accuracy of protein synthesis. How do bacterial and eukaryotic release factors ensure accurate termination? How do mRNA modifications affect the accuracy of bacterial protein synthesis?

Bacterial release factors 1 (RF1) and 2 (RF2) are proteins that recognize the stop codons of mRNA and catalyze the release of a synthesized protein chain from the ribosome. It has been proposed that RFs ensure accurate termination by binding to the ribosome in an inactive, compact conformation and acquire a catalytically active, extended conformation only after recognizing a correct stop codon. However, the native compact conformation was too short-lived to be captured by conventional structural methods. We have developed a fast-kinetics approach for determining when the RFs are in a compact conformation on the ribosome and then used time-resolved cryogenic electron microscopy to capture the compact conformations of native RF1 and RF2 bound to a stop codon. We have also measured the effect of eukaryotic release factor 3 (eRF3) on the rate and accuracy of peptide release by eukaryotic release factor 1 (eRF1) in a yeast (Saccharomyces cerevisiae) in vitro translation system.

Modifications of mRNA nucleotides are post-transcriptional regulators of gene expression, but little is known about their role in protein synthesis. We have studied the effect on accuracy of protein synthesis by two of these modifications: 2’-O-methylation and N6-methylation of adenosine. 2’-O-methylation greatly reduced the maximal rate (kcat) and efficiency (kcat/Km) of cognate (correct) codon reading by decreasing the initial GTPase activity in elongation factor Tu and enhancing proofreading losses of cognate aminoacyl-tRNAs. Remarkably, N6-methylation reduced the efficiency of codon reading by cognate aminoacyl-tRNAs and release factors, leaving the efficiency of the corresponding non-cognate reactions much less affected.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 47
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1814
Keywords
Ribosome, Protein synthesis, Translation, Accuracy, Release factor, Termination, mRNA modifications
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382490 (URN)978-91-513-0667-4 (ISBN)
Public defence
2019-06-04, A1:111a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-05-10 Created: 2019-04-26 Last updated: 2019-06-17

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