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Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The histidine (H) residue at the apex of switch II is conserved in all translational GTPases. Thishistidine (H92) in elongation factor G (EF-G) has been implicated in GTP hydrolysis andinorganic phosphate (pi) release similar to H85 in elongation factor-Tu (EF-Tu). Mutagenesis ofH92 to alanine (A) and glutamic acid (E) showed different degrees of defect in different steps ofelongation. While H92A was ~7 times slower than wild type EF-G in ribosome mediated GTPhydrolysis, it was 100 times slower in both pi release and tRNA translocation. The H92E mutant,on the other hand, was 100 times slower in all these steps. Both mutants were significantlydefective (~1000 times slower) in tripeptide formation that which requires dissociation of EF-Gfrom the post-translocation state. Thus, our results indicate that GTP hydrolysis takes place priorto tRNA translocation, whereas Pi release occurs probably after or independent of thetranslocation step. Since translocation involves back ratcheting of the ribosomal subunits ourresults suggest that there is a cross-talk between GTP hydrolysis by EF-G and ribosomal subunitrotation. We further confirm that Pi release is essential for the next round of elongation.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-207882OAI: oai:DiVA.org:uu-207882DiVA: diva2:650221
Available from: 2013-09-20 Created: 2013-09-20 Last updated: 2017-01-25
In thesis
1. Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria
Open this publication in new window or tab >>Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1079
Keyword
Ribosome, Elongation factor-G, FusB, GTP, Staphylococcus aureus, Escherichia coli and Fusidic acid
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-207924 (URN)978-91-554-8761-4 (ISBN)
Public defence
2013-10-25, B21, BMC, Husargatan, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-10-04 Created: 2013-09-20 Last updated: 2014-01-23

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Koripella, Ravi KiranHolm, MikaelSanyal, Suparna

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