| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 88 === This study is aimed to explore the functional regions of the Bacillus subtilis sA factor. Seven mutants with deletion at the N-terminus and four mutants with deletion at the C-terminus of σA factor were studied for their biochemical properties. The in vitro transcription activity of the reconstituted RNA polymerase holoenzyme containing each of the truncated sA factors varied significantly, depending on the position and numbers of amino acid being deleted. All the four C-terminally truncated sA were inactive. Only SND73-sA and SND94-sA, among the seven mutants with deletion at the N-terminus, had transcription activities comparable to that of the wild-type counterpart after reconstitution with core RNA polymerase. Mechanisms leading transcription inactivation of the truncated sA-RNA polymerase can be categorized into two groups. SCD207-sA, SCD44-sA, and SCD79-sA belong to the first group, of which the activity loss was attributed to the inability of core association; whereas SND26-sA, SND52-sA, and SND129-sA belong to the second group, of which the reconstituted RNA polymerase holoenzymes have lost their promoter-binding activity. The SND94-sA factor had only 50% of the transcription activity of the wild-type counterpart. However, different from the properties of the above-mentioned two groups, it possesses a 5-fold higher activity in core association comparing with the wild type, and a 1.5-fold higher affinity for Φ29 phage G3b promoter after association with core. The decrease in abortive transcription activity of the SND94-sA-RNA polymerase suggests that it is inefficient in triggering the formation of open complex. Other important observations needed to be mentioned are that the most C-terminal part of sA plays an essential role in core binding and that the amino acids at the most N-terminal part of sA determine the promoter DNA-binding activity of the RNA polymerase holoenzyme. Probably, the N-terminal 26 amino acids would induce a conformational change of sA, which enables the holoenzyme to bind promoter DNA. Furthermore, the SND73-sA, although lacks the N-terminal 73 amino acids, remains active in vitro and possesses several essential functions required for transcription initiation. It seems that the N-terminal 73 amino acids of sA participate in the regulation of genes controlled by sA in B. subtilis.
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