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On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
2015 (engelsk)Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 9, s. 1848-1860Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s(-1) with an apparent pK(a) value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others.

sted, utgiver, år, opplag, sider
2015. Vol. 427, nr 9, s. 1848-1860
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-255073DOI: 10.1016/j.jmb.2015.01.007ISI: 000353929400006PubMedID: 25619162OAI: oai:DiVA.org:uu-255073DiVA, id: diva2:821760
Forskningsfinansiär
Knut and Alice Wallenberg FoundationSwedish Research CouncilTilgjengelig fra: 2015-06-15 Laget: 2015-06-12 Sist oppdatert: 2019-04-26bibliografisk kontrollert
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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