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Bradley Johnson

Advisor: Alicia Dombroski, Ph.D.

The sigma subunit of eubacterial RNA polymerase (RNAP) is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of bacterial gene expression can be achieved by modulating sigma factor activity. The Bacillus subtilis sporulation sigma factor, sigmaK, controls gene expression of the late sporulation regulon. SigmaK is synthesized as an inactive precursor protein, pro-sigmaK, with a 20 amino acid pro sequence. Proteolytic processing of the pro sequence produces the active form, sigmaK, which is able to bind to the core subunits of RNAP to direct gene expression. Thus, the pro sequence renders sigmaK inactive in vivo. After processing, the amino terminus of sigmaK consists of region 1.2, which is conserved among various sigma factors. To understand the role of the amino terminus of sigmaK, namely the pro sequence and region 1.2, mutagenesis of both regions was pursued. NH2-terminal truncations of pro-sigmaK were constructed to address how the pro sequence silences sigmaK activity. The work described here shows that the pro sequence inhibits the ability of sigmaK to associate with the core subunits and that a deletion of only six amino acids of the pro sequence is sufficient to activate pro-sigmaK for DNA binding and transcription initiation to levels similar to sigmaK. Additionally, site directed mutagenesis was used to obtain single amino acid substitutions in region 1.2 to address the role of region 1.2 in sigmaK transcriptional activity. Two mutations were isolated, converting a lysine (K) to an alanine (A) at position three, and an asparagine (N) to a tyrosine (Y) at position five, both of which alter the efficiency of transcription initiation by RNAP containing the mutant sigmaKs. Surprisingly, sigmaKK3A increased transcript production when compared to wild type. This increase is due to improvement in DNA affinity and increased stability of RNAP-DNA promoter open complexes. SigmaKN5Y showed a decrease in transcription activity that is related to defects in the ability of RNAP to make the transition from the closed to open RNAP-DNA complex. Results of both the pro sequence and region 1.2 analyses indicate that the amino terminus of sigmaK is important for transcription activity and this work adds to the increasing body of evidence that the amino termini of many sigma factors modulate transcription initiation by RNAP.

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Research Info

The Role of the Amino Terminus of Bacillus subtilis SigmaK in Transcription Initiation