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Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
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.
2008 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 5, 1194-1209 p.Article in journal (Refereed) Published
Abstract [en]

Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

Place, publisher, year, edition, pages
2008. Vol. 70, no 5, 1194-1209 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-107026DOI: 10.1111/j.1365-2958.2008.06472.xISI: 000261070300012PubMedID: 18990188OAI: oai:DiVA.org:uu-107026DiVA: diva2:227577
Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2011-11-10Bibliographically approved
In thesis
1. EF-Tu and RNase E: Essential and Functionally Connected Proteins
Open this publication in new window or tab >>EF-Tu and RNase E: Essential and Functionally Connected Proteins
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme.

We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode.

RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 863
bacterial growth, translation, EF-Tu, RNase E, mRNA, RNA degradation
National Category
Research subject
Molecular Cellbiology; Microbiology
urn:nbn:se:uu:diva-159682 (URN)978-91-554-8179-7 (ISBN)
Public defence
2011-11-24, B21, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2011-11-03 Created: 2011-10-06 Last updated: 2011-11-10Bibliographically approved

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Hammarlöf, Disa LHughes, Diarmaid
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