Study On Archaeal RNA Polymerase Assembly And Dissection Of The Core Elements On An Archeal Promoter

In this study, RNA polymerase (RNAP) subunit gene rpoL was cloned into pSeSD and transformed into Sulfolobus islandicus for expression. A protein complex was purified by nickel affinity purification, followed by gel filtration. The complex was confirmed to be RNAP by mass spectra. The RNAP complex was treated with2M/4M/6M urea, in combination with1M/2M/3M NaCl to determine the interactions among the subunits. The result indicated RpoL strongly bond to RpoB", RpoA’ and RpoA" rather than other subunits. It was presumed that the RNAP subunits assembly process differed from its eukaryotic partner. In Sulfolobus the "L-B"-A’-A"" platform was assembled first, and other subunits were loaded after that. Secondly, the general transcriptional factor (TBP and TFB1) in Sulfolobus were expressed in E. coli cells. In EMS A, the purified RNAP, TBP and TFB1were used to determine the binding efficiencies between transcriptional machinery and test promoters (araS basal promoter and T6BRE hybrid promoter). The EMSA result indicated Sulfolobus transcriptional machinery bond to T6BRE stronger than to araS promoter. The in vitro result confirmed that the defective BRE in araS basal promoter hindered its promoter activity.Based on vector pSeSD, a tandem affinity purification vector and a dual-host expression vector were constructed. It was confirmed that the tandem affinity vector which carried Strep tag and His tag could increase the specificity in co-purification assay and dual host expression vector could utilize T7promoter in E. coli and araS promoter in Sulfolobus to control the expression of target gene.Moreover, the trans-membrane protein expression vector based on pSeSD was constructed. The out-membrane protein a-Amylase was used as the carrier protein, and its gene was cloned into pSeSD. The result indicated fusion protein was transferred out of host cells using lacS as the reporter gene.Finally, the promoter element of single stranded DNA binding protein (ssb) gene was dissected by reporter gene system. The result indicated, although BRE was defective, the ssb basal promoter showed strong activity. And it suggested a downstream activation sequence (DAS) might existed.Taking all together, this work provided good materials for identification of Sulfolobus RNAP subunits assembly, characterization of ssb DAS DNA element and characterization of protein interactions in Sulfolobus by co-purification method.