title: Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein.
abstract: 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 σ-factors (σ(70), σ(S), σ(54), σ(32), σ(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σ(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 σ(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.
authors.affiliation: Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Stec-Dziedzic
authors.forename: Robert
authors.initials: R
authors.lastname: Lyżeń
authors.forename: Eleonore
authors.initials: E
authors.lastname: Skärfstad
authors.forename: Victoria
authors.initials: V
authors.lastname: Shingler
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Szalewska-Pałasz
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074434:RapA protein, E coli; D012321:DNA-Directed RNA Polymerases