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Influences of a mutated translation initiation factor IF1 or kasugamycin on  Escherichia coli gene expression
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Leif Isaksson's)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Leif Isaksson's)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
(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.

Keyword [en]
E. coli; initiation factor IF1; kasugamycin; 2D-PAGE; initiation region
National Category
Microbiology in the medical area Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-39299OAI: oai:DiVA.org:su-39299DiVA: diva2:319359
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2012-01-16Bibliographically approved
In thesis
1. Studies on Translation Initiation and trans-Translation
Open this publication in new window or tab >>Studies on Translation Initiation and trans-Translation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

 Translation initiation factor 1 (IF1) is an essential bacterial protein, without any clearly defined function in translation initiation. Same point mutants in this protein exhibit cold-sensitivity. In addition, a sequence specific increase in test protein expression has been observed for these mutants. Here in Paper I we analyze the parts of mRNA translation initiation region (TIR) involved in this effect, showing that Shine-Dalgarno sequence (SD), upstream enhancer region as well as linker between the initiation codon and SD are important. The similarity in action between the mutated IF1 and the antibiotic kasugamycin leads us to the suggestion that it occurs during the recently discovered proof-reading stage after the subunit joining step in the translation initiation. Deletion of yggJ and cspA genes involved in mRNA translation partially suppresses the cold-sensitivity phenotype of the R40D IF1 mutant. Deletion of the bipA gene confers even higher suppression confirming the previously assigned function of this protein in initiation of translation. In Paper II of this thesis proteome analysis of the IF1 mutation and kasugamycin action reveals some interesting features, such as increase in S6 polyglutamylation in the IF1 mutant cells or high level of GroEL-GroES protein in the cells treated with kasugamycin. A subset of genes having a similar TIR-specific increase in the expression under both conditions confirms previously made observations. Finally, in the Paper III we use cryo-electron tomography as well as chemical probing and computational modeling to address the question, how tmRNA moves on the ribosome during trans-translation. We observe that the tmRNA pseudoknots remain intact, while the mRNA part moves in the mRNA channel, allowing translation elongation to proceed.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology,Stockholm university, 2010. 96 p.
Keyword
E. coli, ribosome, IF1, tmRNA, trans-translaton
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-39304 (URN)978-91-7447-099-4 (ISBN)
Public defence
2010-06-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.Available from: 2010-05-27 Created: 2010-05-17 Last updated: 2010-08-04Bibliographically approved
2. 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.
Keyword
Escherichia coli, translation, ribosome, IF1, RF1, kasugamycin, tRNA
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
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)
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: 2012-01-24Bibliographically approved

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