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2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation
Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
SLAC Natl Accelerator Lab, Stanford PULSE Inst, Menlo Pk, CA USA.;SLAC Natl Accelerator Lab, Biosci Div, Menlo Pk, CA USA..ORCID iD: 0000-0002-9135-5397
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
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2018 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 3, p. 208-216Article in journal (Refereed) Published
Abstract [en]

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2 '-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2 '-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2 '-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2018. Vol. 25, no 3, p. 208-216
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-350295DOI: 10.1038/s41594-018-0030-zISI: 000426704000006PubMedID: 29459784OAI: oai:DiVA.org:uu-350295DiVA, id: diva2:1204716
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilAvailable from: 2018-05-09 Created: 2018-05-09 Last updated: 2019-04-26
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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