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N6-methyladenosines in mRNA have profound effects on the accuracy of codon reading by tRNAs and peptide release factors
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi. Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA. Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA, USA.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-382013OAI: oai:DiVA.org:uu-382013DiVA, id: diva2:1307003
Tilgjengelig fra: 2019-04-25 Laget: 2019-04-25 Sist oppdatert: 2019-04-26
Inngår i avhandling
1. Accuracy of protein synthesis and its tuning by mRNA modifications
Åpne denne publikasjonen i ny fane eller vindu >>Accuracy of protein synthesis and its tuning by mRNA modifications
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

 

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 47
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1814
Emneord
Ribosome, Protein synthesis, Translation, Accuracy, Release factor, Termination, mRNA modifications
HSV kategori
Forskningsprogram
Biologi med inriktning mot molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-382490 (URN)978-91-513-0667-4 (ISBN)
Disputas
2019-06-04, A1:111a, BMC, Husargatan 3, Uppsala, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-05-10 Laget: 2019-04-26 Sist oppdatert: 2019-06-17

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