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Accuracy of protein synthesis and its tuning by mRNA modifications
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
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 [en]
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: urn:nbn:se:uu:diva-382490ISBN: 978-91-513-0667-4 (print)OAI: oai:DiVA.org:uu-382490DiVA, id: diva2:1307157
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
List of papers
1. On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
Open this publication in new window or tab >>On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, p. 1848-1860Article in journal (Refereed) 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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-255073 (URN)10.1016/j.jmb.2015.01.007 (DOI)000353929400006 ()25619162 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2015-06-15 Created: 2015-06-12 Last updated: 2019-04-26Bibliographically approved
2. The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy
Open this publication in new window or tab >>The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy
Show others...
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382011 (URN)10.1038/s41467-019-10608-z (DOI)000471224100009 ()31189921 (PubMedID)
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
3. 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation
Open this publication in new window or tab >>2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation
Show others...
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
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-350295 (URN)10.1038/s41594-018-0030-z (DOI)000426704000006 ()29459784 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2018-05-09 Created: 2018-05-09 Last updated: 2019-04-26
4. N6-methyladenosines in mRNA have profound effects on the accuracy of codon reading by tRNAs and peptide release factors
Open this publication in new window or tab >>N6-methyladenosines in mRNA have profound effects on the accuracy of codon reading by tRNAs and peptide release factors
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382013 (URN)
Available from: 2019-04-25 Created: 2019-04-25 Last updated: 2019-04-26

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