Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
EF-Tu and RNase E: Essential and Functionally Connected Proteins
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 863
Keyword [en]
bacterial growth, translation, EF-Tu, RNase E, mRNA, RNA degradation
National Category
Microbiology
Research subject
Molecular Cellbiology; Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-159682ISBN: 978-91-554-8179-7 (print)OAI: oai:DiVA.org:uu-159682DiVA: diva2:446236
Public defence
2011-11-24, B21, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-11-03 Created: 2011-10-06 Last updated: 2011-11-10Bibliographically approved
List of papers
1. Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
Open this publication in new window or tab >>Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-107026 (URN)10.1111/j.1365-2958.2008.06472.x (DOI)000261070300012 ()18990188 (PubMedID)
Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2017-12-13Bibliographically approved
2. Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
Open this publication in new window or tab >>Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, e90486- p.Article in journal (Other academic) Published
Abstract [en]

Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

Keyword
tufA; ppGpp; RelA; Salmonella enterica; growth regulation
National Category
Microbiology
Research subject
Microbiology; Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-159663 (URN)10.1371/journal.pone.0090486 (DOI)000332396200210 ()
Note

Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.

Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
3. Temperature-sensitive mutants of RNase E in Salmonella enterica
Open this publication in new window or tab >>Temperature-sensitive mutants of RNase E in Salmonella enterica
2011 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, 6639-6650 p.Article in journal (Refereed) Published
Abstract [en]

RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

National Category
Natural Sciences Microbiology
Identifiers
urn:nbn:se:uu:diva-159661 (URN)10.1128/JB.05868-11 (DOI)000296795600023 ()21949072 (PubMedID)
Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
4. Extragenic suppressors of RNase E ts mutants
Open this publication in new window or tab >>Extragenic suppressors of RNase E ts mutants
(English)Manuscript (preprint) (Other academic)
Abstract [en]

RNase E is an essential endoribonuclease and plays a central role in regulating mRNA levels and stable RNA activity in the bacterial cell. Previous studies of RNA half-life and processing in strains carrying rne mutations have shown that it is the catalytic half of RNase E that is essential for bacterial growth, but have not identified a specific reason for this essentiality. In this study we have used two ts mutations in the catalytic region of RNase E (rne-6 and rne-9) from Salmonella as tools to select and screen for extragenic suppressors of the temperature-sensitive phenotype. We reasoned that identifying extragenic suppressors might give information on the essential function of RNase E. 15 independent extragenic suppressors were isolated and mapped to three different loci on the Salmonella chromosome: rpsA (encoding ribosomal protein S1); vacB (encoding RNase R); and within and neighbouring the ORFs STM1551/1550, putatively encoding a toxin-antitoxin system similar to RelBE from E. coli. Each suppressor mutation could cross-suppress the ts phenotypes of rne-6 and rne-9 and each suppressor mutation alone was viable in a wild-type background. We discuss a model where at the non-permissive temperature an excess of mRNA (including defective species) may trap ribosomes non-productively, reducing the rate of protein synthesis and growth. Accordingly the rpsA mutation may suppress the ts phenotype by reducing the rate of translation initiation, and by default increasing the probability that residual RNase E activity turns over mRNA. The vacB mutations may expand the substrate range of RNase R allowing it to more efficiently substitute for poorly active RNase E in degrading mRNA. Finally, the mutations in the STM1551 region may increase the amount of RelE-like toxin and thereby increase the rate of mRNA turnover. This model makes predictions which can be experimentally tested.

Keyword
rpsA; vacB; RNase R; RelBE; RNase E; mRNA turnover
National Category
Microbiology
Research subject
Microbiology; Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-159662 (URN)
Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2011-11-10

Open Access in DiVA

fulltext(1896 kB)3598 downloads
File information
File name FULLTEXT01.pdfFile size 1896 kBChecksum SHA-512
569e3aaa5164ece499f32b020f5472da96214d744cba94fe90483fa907c73824133e94452172be1f73a2c62fb530e5478610240922ad2c7dc925a0f9a887d2dd
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Hammarlöf, Disa L.
By organisation
Microbiology
Microbiology

Search outside of DiVA

GoogleGoogle Scholar
Total: 3598 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 910 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf