Promoter Functional Study On The RM07 DNA Fragment From Halophilic Archaea In Three Domains Of Life

Using the Escherichia coli promoter probe vector pKK232-8, a 492 bp DNA fragment, designated RM07, was isolated from the chromosomal DNA of the halophilic Archaea, Halobacterium halobium, and was shown to confer promoter activity in Escherichia coli. Sequence analysis revealed that RM07 fragment contained the typical -35 and -10 box consensus sequences of bacterial promoter as well as the characteristic sequence of archaeal promoter-DPE(distal promoter element).Using the bgaH, dhfr, cat and neo gene as the reporter genes, various RM07-reporter gene fusion plasmids were constructed and transformed into Haloferax volcanii, Escherichia coli and Saccharomyces cerevisiae respectively. Through determining the enzymatic activity, detecting the antibiotic resistance level and RT-PCR analysis, it was confirmed that RM07 conferred promoter activity in all three domains of life: Archaea (Haloferax volcanii), Bacteria (Escherichia coli) and Eukarya (Saccharomyces cerevisiae).Deletion analysis of RM07 was performed in Haloferax volcanii, Escherichia coli and Saccharomyces cerevisiae. The recombinant plasmids with different deletion fragments of RM07 inserted upstream of reporter gene were constructed. Through detecting the promoter activity of various deletion fragments of RM07, the important functional regions within RM07 which could influence the promoter activity were identified. The results revealed that the 40bp region containing the typical -35 and -10 box sequences of bacterial promoters was the important functional region responsible for the basal promoter activity of RM07 in Escherichia coli. The characteristic sequence of archaeal promoter-DPE(distal promoter element) was the functional element necessary for thepromoter activity of RM07 in both Archaea (Haloferax volcanif) and Eukarya(Saccharomyces cerevisiae). The promoter functional elements (-35 , -10 box andarchaeal DPE) were related.Site-directed mutagenesis of RM07 was performed in Escherichia coll.Different nucleotide mutations had different effect on the promoter activity of RM07. The critical nucleotide responsible for the promoter function of RM07 in Escherichia colt was determined precisely. The possible transcription initial nucleotide site was also identified. The promoter activity of RM07 in Escherichia coli was improved greatly by modifying the nucleotide component in RM07.In this study, the important thermochemistry research method microcalorimetry was successfully applied in studying the structure and function of promoter in Escherichia coli. The research results of microcalorimetry further confirmed that RM07 could confer promoter activity in Escherichia coli. The different effects of various nucleotide mutations on the promoter activity of RM07 were detected by means of microcalorimetric method. The results were consistent with the results gotten by the traditional biological method. Our work also provided a more sensitive and easily-performed novel method combining the chemical and biological technique for studying the promoter function.