Abstract

This thesis describes mechanism of the regulation of the transcription of the rpoE gene encoding an essential RNA polymerase subunit in Escherichia coli. The RpoE regulates extracytoplasmic stress response regulon and is required to initiate transcription of genes, whose products are involvedin the folding of periplasmic proteins and synthesis and transport of outer membrane components. The transcriptional regulation of the rpoE gene has not been fully understood. Initially two promoters were identified, out of which the downstream promoter is positively regulated by RpoE. In this study, it was shown that transcription from upstream region is sustained under several stress and non-stress conditions such as entry into the stationary phase or high osmolarity or cold shock or treatment with antibiotics like polymyxin B or defects in lipopolysaccharide (LPS) assembly and its biosynthesis. Such multiple divergent regulations of the upstream promoter suggested that there could be more than one transcription start site located within this region. Indeed, mapping of 5’ ends of the rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to the promoter regulated by RpoE. Experiments showed that three major promoters, P2, P3 and P4, are recognized by different sigma factors (RpoN, RpoD and RpoS) and these promoters are activated in response to unique signals. The RpoN-dependent P2 promoter was shown to require the QseF as an activator. Analysis of trans acting mutations revealed that LPS defects caused the induction of transcription from the RpoD-regulated rpoEP3 promoter, revealing the mechanism of sensing of LPS alterations in E. coli. This promoter is also positively regulated by the Rcs system and by the global regulator CRP. Furthermore, two non-coding small regulatory RNAs (sRNAs) were identified. One of them, SlrA, was shown to act in trans and repress the synthesis of most abundant protein Lpp and prevent hyperinduction of RpoE, when LPS biosynthesis is compromised in the absence of essential LapB protein. The second sRNA identified was shown to regulate LPS biosynthesis by sequestering RfaH transcriptional factor. Furthermore, mutagenesis revealed essential amino acid residues that are required for the function of LPS assembly factor LapB.

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