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Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature-sensitive phenotype caused by mutant release factor 1
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2009 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 5, 1604-9 p.Article in journal (Refereed) Published
Abstract [en]

In Escherichia coli, release factor 1 (RF1) is one of two RFs that mediate termination; it specifically recognizes UAA and UAG stop codons. A mutant allele, prfA1, coding for an RF1 that causes temperature-sensitive (Ts) growth at 42 degrees C, was used to select for temperature-resistant (Ts(+)) suppressors. This study describes one such suppressor that is the result of an IS10 insertion into the cysB gene, giving a Cys(-) phenotype. CysB is a transcription factor regulating the cys regulon, mainly as an activator, which explains the Cys(-) phenotype. We have found that suppression is a consequence of the lost ability to donate sulfur to enzymes involved in the synthesis of thiolated nucleosides. From genetic analyses we conclude that it is the lack of the 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) modification of the wobble base of tRNA(Glu), tRNA(Lys), and/or tRNA(Gln) that causes the suppressor phenotype.

Place, publisher, year, edition, pages
2009. Vol. 191, no 5, 1604-9 p.
National Category
Natural Sciences
URN: urn:nbn:se:su:diva-31974DOI: 10.1128/JB.01485-08ISI: 000263503600027PubMedID: 19103926OAI: diva2:279087
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2012-01-16Bibliographically approved
In thesis
1. Studies on Translation Initiation and Termination in Escherichia coli
Open this publication in new window or tab >>Studies on Translation Initiation and Termination in Escherichia coli
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Translation initiation factor 1 (IF1) has been shown to be an RNA chaperone. In order to find functional interactions that IF1 may have with rRNA, we have isolated second-site suppressors of a cold-sensitive IF1 mutant. Joining of the ribosomal subunit seems to be affected in the IF1 mutant strain and the suppressive effect is a consequence of decreasing the available pool of mature 50S subunits. The results serve as additional evidence that IF1 is an RNA chaperone and that final maturation of the ribosome takes place during translation initiation. In this study we have also investigated the effect of a cold-sensitive mutant IF1 or kasugamycin addition on gene expression using a 2D gel electrophoresis technique. The effect is much more dramatic when cells are treated with kasugamycin compared to mutant IF1. The ybgF gene is uniquely sensitive to the IF1 mutation as well as the addition of kasugamycin. This effect on the native gene could be connected with some property of the TIR sequence of ybgF and supports the notion that kasugamycin addition and the IF1 cold-sensitive mutation have a similar TIR-specific effect on mRNA translation. Finally we have isolated a suppressor of a temperature-sensitive mutation in ribosomal release factor 1 (RF1) to shed more light on the translation termination process. The suppressor mutation is linked to an IS10 insertion into the cysB gene and results in a Cys- phenotype. Our results suggest that suppression of the thermosensitive growth is a consequence of the mnm5s2U hypomodification of certain tRNA species. The ability of mnm5s2U hypomodified tRNA to induce frameshifting may be responsible for the suppression mechanism and it supports the hypothesis that modified nucleosides in the anticodon of tRNA act in part to prevent frameshifting by the ribosome.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 58 p.
Escherichia coli, translation, ribosome, IF1, RF1, kasugamycin, tRNA
National Category
Natural Sciences
Research subject
Molecular Genetics
urn:nbn:se:su:diva-69954 (URN)978-91-7447-388-9 (ISBN)
Public defence
2012-02-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 13:00 (English)
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.Available from: 2012-02-02 Created: 2012-01-16 Last updated: 2012-01-24Bibliographically approved

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Isak, Georgina
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