Functional analysis of the RNA polymerase binding protein RbpA in Streptomyces coelicolor

In all organisms genes are switched on or off in response to environmental, developmental or physiological cues. In bacteria, the key step in the control of gene expression is transcription initiation. Transcription initiation proceeds via several steps including the binding of an 'RNA polymerase' complex to a 'promoter' sequence upstream of the gene, opening of the double-stranded DNA to expose the single-stranded template, and synthesis of messenger RNA (mRNA) on the template strand (mRNA is subsequently decoded by ribosomes in the production of proteins). The frequency of transcription initiation is controlled by regulatory proteins, most of which bind to DNA in the promoter region, often also interacting with RNA polymerase. In addition, several proteins interact stably with RNA polymerase to modulate its activity without binding to DNA. RbpA is an RNA polymerase binding protein in the industrially important antibiotic-producing bacterium Streptomyces coelicolor. This organism is the model representative of a large family of bacteria that produce the majority of antibiotics in use today. Our preliminary data suggests that RbpA regulates the production of ribosomes, by activating expression of the RNA component of these protein factories. In all organisms ribosome production is very carefully regulated in response to protein synthesis needs and nutrient availability, leading to a close correlation between ribosome numbers and growth rate. The importance of RbpA is underlined by the finding that mutants that lack this protein grow slowly. Another interesting consequence of removing RbpA is increased sensitivity to rifampicin, an antibiotic that binds RNA polymerase and blocks transcription initiation. The overall aim of this project is to understand how RbpA regulates ribosomal RNA synthesis. This research is important because antibiotic production in the Streptomyces bacteria is linked to growth rate: antibiotics are usually only produced when the organism stops replicating. RbpA-related proteins are also found in related pathogenic organisms, including Mycobacterium tuberculosis, which claims ~2 million lives annually. Rifampicin is a front-line drug used to combat this pathogen, although drug-resistant mutants are an increasing problem. Therefore the outcomes of this research will be relevant to researchers investigating both growth rate and mechanisms of rifampicin resistance in this organism.

RbpA is an RNA polymerase (RNAP)-binding protein that occurs in the actinomycete family of bacteria and is regulated by the disulphide stress-response sigma factor, SigR, in Streptomyces coelicolor. rbpA null mutants exhibit a slow-growth phenotype and increased sensitivity to rifampicin. Reconstitution of RNAP with purified RbpA revealed that RbpA stimulates transcription from the rRNA operon promoter rrnDp3, and partially protects it from the inhibitory effect of rifampicin. Our data suggests that RbpA regulates rRNA synthesis, and thereby growth rate, using a novel mechanism; our overall aim is to understand the function of RbpA and its mechanism of action. We propose to: (1) use purified components and coelution assays to determine whether RbpA interacts with core RNAP, total holoenzyme, or specific sigma-containing holoenzymes; (2) conduct a series of in vitro transcription and binding experiments aimed at dissecting the mechanism of RbpA action at rrnDp3; (3) use rrnDp3 transcription fusions and western analysis to monitor RbpA activity and levels during different growth phases and in response to disulphide stress; (4) investigate whether the positive role of RbpA in rRNA synthesis is the primary cause of the slow-growth and rifampicin-sensitive phenotypes of rbpA null mutants.