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Studies on Translation Initiation and trans-Translation
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Leif Isaksson)
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 [en]
E. coli, ribosome, IF1, tmRNA, trans-translaton
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-39304ISBN: 978-91-7447-099-4 (print)OAI: oai:DiVA.org:su-39304DiVA: diva2:319483
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
List of papers
1. Structural features of the tmRNA-ribosome interaction
Open this publication in new window or tab >>Structural features of the tmRNA-ribosome interaction
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2009 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 15, no 12, 2312-2320 p.Article in journal (Refereed) Published
Abstract [en]

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

Place, publisher, year, edition, pages
RNA Society, 2009
Identifiers
urn:nbn:se:su:diva-31967 (URN)10.1261/rna.1584209 (DOI)000272169000020 ()19861420 (PubMedID)
Note
9 authorsAvailable from: 2009-12-01 Created: 2009-12-01 Last updated: 2010-08-04Bibliographically 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.

Keyword
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: 2012-01-16Bibliographically approved
3. Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin
Open this publication in new window or tab >>Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin
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2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 11, 2428-2439 p.Article in journal (Refereed) Published
Abstract [en]

Translation initiation factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation initiation on the ribosome remains unclear, even though the factor has several known functions, one of them being RNA chaperone activity. In this study, we analyzed translational gene expression mutant variants of IF1 with amino acid substitutions, R40D and R69L, using two different reporter gene systems. The strains with them mutant IF1 gave higher reporter gene expression than the control strain. The extent of this effect was dependent on the composition of the translation initiation region. The Shine–Dalgarno (SD) sequence, AU-rich elements upstream of the SD sequence and the region between the SD sequence and the initiation codon are important for the magnitude of this effect. The data suggest that the wild-type form of IF1 has a translation initiation region-dependent inhibitory effect on translation initiation. Kasugamycin is an antibiotic that blocks translation initiation. Addition of kasugamycin to growing wild-type cells increases reporter gene expression in a very similar way to the altered IF1, suggesting that the and kasugamycin affect some related step in translation initiation. Genetic knockout of three proteins (YggJ, BipA, and CspA) that are known to interact with RNA causes partial suppression of the IF1-dependent cold sensitivity.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2010
Keyword
bipA; cspA; infA(IF1); kasugamycin; yggJ
National Category
Biochemistry and Molecular Biology
Research subject
Microbiology; Molecular Biology
Identifiers
urn:nbn:se:su:diva-39289 (URN)10.1111/j.1742-4658.2010.07657.x (DOI)000277800600006 ()
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12Bibliographically approved

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