The Importance of Amino Acid Residues at the C-terminus of Region 1.1 to the Structural and Functional Properties of Bacilis subtilis sigA factor

• Main Author: 許信賢
• Other Authors: 張邦彥
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/t8m7qz

碩士 === 國立中興大學 === 生物化學研究所 === 90 === Abstract The sigma (σ) subunit of prokaryotic RNA polymerase is essential for the recognition of promoter DNA and the initiation of transcription. Our previous data have revealed that the truncated A factor of B. subtilis, with N-terminal 26, 52, 73, 94, or 103 amino acids being deleted, is as functional as its wild-type counterpart after reconstitution with core RNA polymerase. However, further N-terminal deletion into residue 106 has resulted in an abrupt reduction in transcription activity of the RNA polymerase harboring the truncated A. This purpose of this study was to investigate the role of amino acid residues, spanning 103 to 106, on the structural and functional properties of A. The experimental data showed that both SND104- and SND105-A-RNA polymerases also significantly reduced their in vitro activities. Since the difference between SND103- and SND104-A was the presence or absence amino acid residue 103 at the N-terminus of the truncated A, it seemed that this amino acid was essential to the maintenance of functional A properties. Single cycle in vitro transcription has been adopted to compare the transcription properties of both the wild-type and N-terminally trancated A-RNA polymerases. The data revealed that SND103-A-RNA polymerase, in comparison with the wild-type one, had a delayed in vitro transcription activity, indicating that at least the efficiency of open complex formation was affected by removal of N-terminal 102 amino acids of A. The defect of delayed transcription was much more pronounced with SND104-A-RNA polymerase under the same condition. Reasons responsible for the defect of SND104-A-RNA polymerase were irrelevant to the core-binding activity of SND104-A and the promoter-binding activity of SND104-A-RNA polymerase as evidenced by gel filtration and gel retardation assays. It was probably a result of inefficient open complex formation of SND104-A-RNA polymerase and G3b promoter or a result of inefficient transition from transcription initiation to transcription elongation. To unravel the mystery resulting in the defect of SND104-A, I compared the amounts of secondary structures possessed by SND104- and SND105-A, which had reduced transcription activity, as well as SND103-A, which possessed normal multiple cycle transcription activity. Interestingly, data from circular dichroism showed that less amount of secondary structure was possessed by the active SND103-A than by the inactive ones. Since a relatively low amount of secondary structures was also observed for the active SND100- or SND102-A comparing with that for SND104-A, it tends to suggest that the amino acid residues located in the junction of regions 1.1 and 1.2 were very critical for the truncated A to possess a correct functional structure.