Identification and Characterization of the Conserved Region 1 of Bacillus subtilis sigmaA Factor

• Main Authors: Liang-Yin Huang, 黃亮尹
• Other Authors: Ban-Yang Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/39565274079948701067

碩士 === 國立中興大學 === 生物化學研究所 === 89 === The sigma subunit of prokaryotic RNA polymerase is essential for promoter recognition and initiation of transcription. Our previous study has shown that the deletion mutant sigmaA factors, SND73- and SND94-sigmaA, are active in transcription after reconstitution with core RNA polymerase; however, SND129-sigmaA is inactive due to the loss of promoter-binding activity of the mutant sigmaA-RNA polymerase. The present study is aimed to explore how much amino acid residues in the N-terminal region of sigmaA are unnecessary for its full in vitro activity, and why further deletion from this amino acid position would cause the loss of transcription activity of the mutant sigmaA factor. Besides, we hope to obtain a series of functional sigmaA factors, which would enable us to study the crystal structure or the structural and functional relationship of sigmaA factor in the future. To fulfill this goal, 25 mutant sigmaA factors, with deletion at the N-terminus and spanning amino acid no. 74 to 127 of sigmaA, were constructed and purified. The functional properties of these mutant sigmaA factors were analyzed in vitro. Our data revealed that deletion of more than 103 amino acids at the N-terminus of sigmaA have resulted in a significant loss of the transcription activity of the reconstituted mutant sigmaA-RNA polymerases. Gel retardation assays indicated that the inactivation of transcription of these mutant sigmaA-RNA polymerases was due to the reduction of promoter-binding activity of the reconstituted holoenzyme. Since no drastic change in the protein conformation was observed among the free SND100-, SND103-, SND106- and SND109-sigmaA factors, and that a more extended deletion at the N-terminus of sigmaA (up to 129 amino acid deletion) would increase the core-binding activity of mutant sigmaA factors, I propose that sigmaA factors with more than 103 amino acids being deleted at the N-terminus do not possess functional conformation when they are associated with core RNA polymerase.