| Microorganisms can utilize different kinds of sugars as carbon source and energy source, e.g. E. coli can utilize lactose and L-arabinose while yeast, the eukaryotic single-cell microorganism, can utilize galactose. The expressions of genes involved in sugar metabolism mentioned above are induced by substrates and highly regulated. In the third domain of life, the hyperthermophile Sulfolobus solfataricus P2 can utilize different sugars, and D-arabinose is one of the favorite sugars which can induce the expression of several genes including the ara operon. The transcription of ara operon is controlled by the araS promoter. So dissection of araS promoter will lead to reveal the mechanism of transcriptional regulation of sugar metabolism and establish the model of transcriptional regulation in Archaea.In order to dissect the cis-acting DNA elements in archaeal promoters, we constructed a reporter gene system for Sulfolobus using lacS as the reporter gene. Archaeal promoters such as araS promoter (including promoter deletion mutants and substitution mutants) were fuesd to the 5'end of lacS gene and the promoter-lacS fusion was inserted into E. coli-Sulfolobus shuttle vector to give the Sulfolobus reporter plasmid. S. islandicus E233S△pyrEF/△lacS was used as the host in this study. After transformation of repoter plasmid into Sulfolobus, the transcription of lacS gene in the transformants was controlled by the fused promoter in different media. Important cis-acting elements were identified according to changes of LacS activity from different transformants with mutated promoter.The results of araS promoter deletion mutants showed the minimal active promoter was from -55 to +4, related to transcription start site. And this promoter region was termed as ParaS·The ara-box located from -49 to -42 was involved in positive control and deletion of half ara-box or whole ara-box resulted inactive promoter mutants in both arabinose and glucose media. Mutations at flanking sequence of ara-box reduced promoter activities significantly. Especially mutations at the 3'sequence reduced promoter activities to 4.1% and 10.7% of ParaS·The promoter mutants with swapping the ara-box of araS promoter for the ara-boxes from ara-induced gene promoters of araDH, araD, dopDH and kdaD showed the similar transcriptional activation mode. The last four nucleotides of araS BRE were not important for araS promoter activity but the first two adjacent to ara-box was essential. And the alignment of BREs from the five ara-induced gene promoters showed no conservation among each other. Mutations in TATA-box indicated the first 6 nucleotides "TTTATA" were essential but the last two AA were not for araS promoter activity. Another essential promoter element was found located from-12 to -7, which was A/T rich, by systematical mutation analysis. -7 and -8 sites were most essential, since they cannot be changed to cytosine, but change AA at -8 and -7 to guanine and thymine only reduced promoter activity slightly. We supposed this element should interact with TFB, so it was termed TFBdn. Meanwhile, an Initiation Element (IE) was identified around transcription start site and mutation at +1,+2 and +4 significatntly reduced lacS expression.After identification of 5 promoter elements in ParaS,a novel transcriptional activation mechanism was characterized. ParaS basal promoter was inactive in all test media in this study and ara-box can activate the inactive basal promoter in all defined media. This indicated ara-box is a general transcriptional activation element in different media. As we know this is the first promoter element acted as general activation sequence in archaea. Hybrid promoter assay indicated the TATA-box and downstream sequence of araS promoter is active and araS BRE is intrinsically inactive. But ara-box immediate upstream of BRE can complement the inactive BRE. This evidence strongly indicated araS promoter was activated via recruitment of TFB by ara-box binding protein. As we know this is a novel transcriptional activation menchanism in archaea.A series of over-expression vectors were constructed based on ParaS and E. coli-Sulfolobus shuttle vector, and His-tagged Mrell and RFC large subunit were over-expressed in S. islandicus using these vectors. SDS-PAGE results indicated the amounts of Mre11 and RFC1 expressed in Sulfolobus were enough for in vitro biochemistry assay and in vivo pull-down assay. RFC small subunit was identified as a novel partner of Mre11 in the in vivo pull-down assay when using Mre11 as bait. If this is the case, the interaction of Mre11 and RFCs will connect DNA replication and repair in archaea.
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