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Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Leif Isaksson)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Anders Nilsson)
Department of Molecular Biology, Aarhus university.
Department of Molecular Biology, Aarhus university.
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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. Vol. 277, no 11, 2428-2439 p.
Keyword [en]
bipA; cspA; infA(IF1); kasugamycin; yggJ
National Category
Biochemistry and Molecular Biology
Research subject
Microbiology; Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-39289DOI: 10.1111/j.1742-4658.2010.07657.xISI: 000277800600006OAI: oai:DiVA.org:su-39289DiVA: diva2:319352
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12Bibliographically 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

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