Studies on BolA and ribonuclease R: two important factors in the control of bacterial gene expression

Dissertation presented to obtain the Ph.D degree in Biology

The cellular concentration of a given RNA is the result of the balance between its synthesis and degradation. Both DNA transcription and RNA decay control the final levels of each protein in the cell. BolA is an Escherichia coli (E. coli) protein, which induces changes in cell morphology when present in high levels. BolA expression is regulated by two different promoters, a sigma 70 (σ70) promoter responsible for the basal levels of this gene in exponential phase and a sigma S (σS) gearbox promoter important in stress situations and stationary phase of bacterial growth. The first objective of this PhD work was to further characterize the expression of the bolA gene. Based on bioinformatic analysis, we have identified the H-NS protein as a putative transcriptional regulator of BolA. H-NS is a relatively small protein, abundant in bacterial cells and is often compared to eukaryotic histones due to its high affinity for DNA. In order to clarify the possible role of H-NS in BolA transcription, we have constructed an hns E. coli mutant. This mutant was compared to the wild type regarding the levels of bolA mRNA transcript and in vitro DNA-protein interaction studies were performed. These experiments allowed us to demonstrate that H-NS is able to down-regulate the levels of bolA mRNA in exponential phase and bind to the bolA promoter region. In addition, the DNA-protein interaction studies revealed that H-NS has a special affinity to the curved bolA promoter region encompassing both bolA1p and bolA2p promoters.(...)

Financial Support from Fundação para a Ciência e Tecnologia (FCT) – Ph.D: grant - SFRH/BD/36027/2007.