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Mechanisms and Inhibition of EF-G-dependent Translocation and Recycling of the Bacterial Ribosome
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.ORCID-id: 0000-0002-1687-7558
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations.

The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2015. , s. 60
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1268
Emneord [en]
Protein synthesis, Elongation factor G, Translocation, Ribosome recycling, Fusidic acid
HSV kategori
Forskningsprogram
Biologi med inriktning mot molekylär bioteknik
Identifikatorer
URN: urn:nbn:se:uu:diva-258990ISBN: 978-91-554-9289-2 (tryckt)OAI: oai:DiVA.org:uu-258990DiVA, id: diva2:844285
Disputas
2015-09-25, B22, BMC, Husargatan 3, Uppsala, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-09-04 Laget: 2015-07-23 Sist oppdatert: 2015-10-01
Delarbeid
1. Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis
Åpne denne publikasjonen i ny fane eller vindu >>Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis
2015 (engelsk)Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 9, s. 1835-1847Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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Identifikatorer
urn:nbn:se:uu:diva-255072 (URN)10.1016/j.jmb.2014.10.027 (DOI)000353929400005 ()25451025 (PubMedID)
Tilgjengelig fra: 2015-06-15 Laget: 2015-06-12 Sist oppdatert: 2018-01-11bibliografisk kontrollert
2. Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
Åpne denne publikasjonen i ny fane eller vindu >>Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
Vise andre…
2015 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 6, s. 3440-3454Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-247496 (URN)10.1074/jbc.M114.611608 (DOI)000349456000020 ()25451927 (PubMedID)
Tilgjengelig fra: 2015-03-19 Laget: 2015-03-19 Sist oppdatert: 2018-01-11bibliografisk kontrollert
3. Complete kinetic mechanism for recycling of the bacterial ribosome
Åpne denne publikasjonen i ny fane eller vindu >>Complete kinetic mechanism for recycling of the bacterial ribosome
2016 (engelsk)Inngår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, nr 1, s. 10-21Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

Emneord
bacterial ribosome recycling; elongation factor G; ribosome recycling factor; translation rate optimization; protein synthesis
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-258988 (URN)10.1261/rna.053157.115 (DOI)000368967600002 ()26527791 (PubMedID)
Forskningsfinansiär
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Tilgjengelig fra: 2015-08-04 Laget: 2015-07-23 Sist oppdatert: 2017-12-04bibliografisk kontrollert
4. Fusidic acid inhibition of EF-G- and RRF-promoted recycling of the bacterial ribosome
Åpne denne publikasjonen i ny fane eller vindu >>Fusidic acid inhibition of EF-G- and RRF-promoted recycling of the bacterial ribosome
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-258989 (URN)
Tilgjengelig fra: 2015-08-04 Laget: 2015-07-23 Sist oppdatert: 2015-10-01

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