Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein

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 (σ70, σS, σ54, σ32, σE) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σE-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 σE-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.

► Pseudomonas putida RapA is a core RNA polymerase-associated protein. ► P. putida RapA activates transcription in multi-round reactions. ► P. putida RapA stimulates transcription dependent on various σ factors. ► σE-pHtrA promoter activity is impaired in P. putida rapA mutant. ► P. putida RapA enhances stability of competitor resistant open complexes.