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Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2013 (English)In: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1829, no 2, 219-230 p.Article in journal (Refereed) Published
Abstract [en]

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of sigma-factors (sigma(70), sigma(S), sigma(54), sigma(32), sigma(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the sigma(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the sigma(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency. 

Place, publisher, year, edition, pages
2013. Vol. 1829, no 2, 219-230 p.
Keyword [en]
RapA, Pseudomonas putida, Transcription factors, RNA polymerase
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Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-67395DOI: 10.1016/j.bbagrm.2012.11.008ISI: 000315063100004OAI: oai:DiVA.org:umu-67395DiVA: diva2:615889
Available from: 2013-04-12 Created: 2013-03-18 Last updated: 2017-12-06Bibliographically approved

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Skärfstad, EleonoreShingler, Victoria

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Skärfstad, EleonoreShingler, Victoria
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Department of Molecular Biology (Faculty of Medicine)
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