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Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2011 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 10, 1745-1756 p.Article 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.

Place, publisher, year, edition, pages
2011. Vol. 278, no 10, 1745-1756 p.
Keyword [en]
Escherichia coli, RNase III, rRNA mutation, rRNA processing, translation
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-68511DOI: 10.1111/j.1742-4658.2011.08099.xISI: 000290169300014OAI: oai:DiVA.org:su-68511DiVA: diva2:473692
Note
3Available from: 2012-01-07 Created: 2012-01-04 Last updated: 2017-12-08Bibliographically 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.
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
2. Studies on the functional interaction of translation initiation factor IF1 with ribosomal RNA
Open this publication in new window or tab >>Studies on the functional interaction of translation initiation factor IF1 with ribosomal RNA
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 44 p.
Keyword
Escherichia coli, ribosome, rRNA mutation
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-75363 (URN)978-91-7447-520-3 (ISBN)
Public defence
2012-05-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 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 3: Manuscript.

Available from: 2012-05-08 Created: 2012-04-17 Last updated: 2013-04-16Bibliographically approved

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Belotserkovsky, Jaroslav M.Isak, Georgina I.Isaksson, Leif A.
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