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Studies on Translation Initiation and Termination in Escherichia coli
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Leif Isaksson)
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. , p. 58
Keywords [en]
Escherichia coli, translation, ribosome, IF1, RF1, kasugamycin, tRNA
National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-69954ISBN: 978-91-7447-388-9 (print)OAI: oai:DiVA.org:su-69954DiVA, id: diva2:478567
Public defence
2012-02-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 13:00 (English)
Opponent
Supervisors
Note

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: 2018-06-21Bibliographically approved
List of papers
1. Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region
Open this publication in new window or tab >>Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region
2011 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 10, p. 1745-1756Article in journal (Refereed) Published
Abstract [en]

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

Keywords
Escherichia coli, RNase III, rRNA mutation, rRNA processing, translation
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-68511 (URN)10.1111/j.1742-4658.2011.08099.x (DOI)000290169300014 ()
Note
3Available from: 2012-01-07 Created: 2012-01-04 Last updated: 2017-12-08Bibliographically approved
2. Influences of a mutated translation initiation factor IF1 or kasugamycin on  Escherichia coli gene expression
Open this publication in new window or tab >>Influences of a mutated translation initiation factor IF1 or kasugamycin on  Escherichia coli gene expression
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The infA (R40D) mutation or the addition of antibiotic kasugamycin to a wild type strain has been suggested to affect the same related step in translation initiation. Two-dimensional gel electrophoresis of radioactively labeled proteins was used to investigate this effect and to gain an overview of the proteins expressed under these conditions. A number of protein spots with increased or decreased expression levels were identified. The most pronounced increased expression in the IF1 mutant strain were the proteins YbgF (> 5 times) and in the relative abundance of the ribosomal protein S6 (rpsF) isoforms.  In the case of kasugamycin treatment strongly increased expression of natural proteins was seen for OppA, GroL, YbgF and several others. Expression of several proteins was affected similarly as a response to either the infA mutation or to the addition of kasugamycin. The translation initiation regions (TIR) comprising a region upstream and downstream of AUG from several of these genes were cloned into the protein A’ reporter gene system for further analysis. TIR sequences of several natural genes that showed elevated expression levels gave a corresponding increase in gene expression as reflected by the protein A’ expression assay system. This is especially true in the case of that the TIR sequence of ybgF gives a strongly increased expression in agreement with the increased expression observed for the native YbgF protein in the infA mutant or upon kasugamycin addition. The results suggest that the TIR region of ybgF has some unique properties that influence its expression at the early translational phase.

Keywords
E. coli; initiation factor IF1; kasugamycin; 2D-PAGE; initiation region
National Category
Microbiology in the medical area Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-39299 (URN)
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2018-01-12Bibliographically approved
3. Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature-sensitive phenotype caused by mutant release factor 1
Open this publication in new window or tab >>Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature-sensitive phenotype caused by mutant release factor 1
2009 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 5, p. 1604-9Article 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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-31974 (URN)10.1128/JB.01485-08 (DOI)000263503600027 ()19103926 (PubMedID)
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved

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