| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 87 === Abstract
The aim of this work was to study the regulation of rpoH gene expression at transcriptional and translational level. The rpoH gene of X. campestris, which encodes an alternative sigma factor (sigma H), had been cloned and sequenced in this laboratory. Northern blot analysis indicated that the amount of rpoH mRNA could be greatly enhanced during 42°C heat shock and the size of the induced mRNA was about 900 nt. In agreement with this, an inverted repeat sequence at downstream of the rpoH gene was proved to function as a transcription terminator. Results of Northern blot and terminator assays suggested that Xc11 rpoH gene has its own promoter and terminator and this gene was thus predicted to be monocistronic. The transcriptional start site of Xc11 rpoH gene was determined to locate at 20 nt (G) and 21 nt (C) preceding the ATG start codon. Sequence similar to consensus sigma E and sigma 70 type promoters were observed at the upstream region of transcriptional start sites. However, no transcript initiated from the putative sigma 70 type promoter was identified by primer-extension.
In order to analyze the promoter activity of Xc11 rpoH gene, the putative promoter region of rpoH was amplified by PCR and cloned into pFY13-9 promoter-probing vector. By measuring the activity of beta-galactosidase, the effects of heat shock and ethanol shock on PrpoH-lacZ gene fusion in Xc11 and E. coli were determined. The results revealed that the putative promoter region has promoter activity at normal growth temperature and could be induced under heat shock and ethanol stress. In addition, the construct with Xc11 sigma E-type promoter was shown to have a higher b-galactosidase activity when transformed into E. coli ER2566 harboring a plasmid with an inducible E. coli sigma E gene than the control strain without the plasmid. In vitro transcription studies demonstrated that Xc11 rpoH promoter could be recognized by E. coli RNA polymerase holoenzyme containing sigma E or sigma 70. However, recognition of Xc11 rpoH promoter by Xc11 RNA polymerase holoenzyme reconstituted with Xc11 sigma 70 or E. coli could only be verified by gel retardation analysis.
The effect of heat shock on the level of rpoH gene expression was further analyzed by Northern blot analysis. A 7-fold increase in the amount of rpoH mRNA was detectable 5 min after heat shock at 42°C and up to 14-fold increase was observed 30 and 60 min after heat shock. However, Western blot analysis indicated that the level of sigma H increase rapidly 5 min after heat shock, reached to its maximal level between 10 and 20 min, and then declined rapidly after 30 min.
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