| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 87 === The events that some of the X. campestris (Xc) promoters, studied by promoter probing vector in vivo, are recognized very poorly by E. coli RNA polymerase (RNAP), lead us to define the characteristic of promoters recognition by Xc RNA polymerase. In order to achieve this study, purification of Xc RNAP was done firstly. Using a step salt gradient of KCl, RNAP holoenzyme (Es70) and core enzyme (E) were partially purified through Heparin-Sepharose CL-6B chromatographic steps. The holoenzyme, which was preliminary isolated at 0.3 M of KCl, was then purified by DEAE A25. Whereas, the core enzyme isolated at 0.4 M was then purified by Bio-Rex 70. The transcriptional activities of these purified RNAPs, as well as the reconstituted Es32 , had been examined by run-off in vitro transcription system using T7A1 and dnaK promoter as template, respectively. With respect to a fixed amount of core polymerase (1 pmol), the amount of sigma 32 required for reconstitution was roughly 3 pmol. Taken together, the results indicated that purified Es70 and Es32 are capable to direct transcription in vitro.
In vitro transcription studies using T7A1 and rpoD promoters and that of Xc and E. coli RNAP under various reaction conditions were performed. Neither Xc nor E. coli RNAP showed differences under different RNAP concentrations and preincubation time factors with T7A1 promoter. In contrast to E. coli enzyme, Xc RNAP was demonstrated to have different behavior under different DNA and NaCl concentrations, and temperature factors. RNAP from Xc11 gave a best fit of transcription under 300 mM of NaCl, 32°C and 2 pmol of DNA template (1:20, RNAP:DNA). Conversely, the conditions for E. coli RNAP is 50 mM of NaCl, 37°C and 2 pmol of DNA template. When Xc11 rpoD promoter was applied as template, the NaCl concentration for Xc RNAP was 100mM and 200 mM for E. coli enzyme. These results revealed that Xc and E. coli RNAP are somewhat different in promoter selectivity.
The promoter specific binding site of Xc RNAP to the rpoD promoter at 28°C was demonstrated by DNase I footprint analysis. The protected region in the non-template strand was from base +18 to -40, which is suggested to be binding site of Xc RNAP open complex. The DNaseI hypersensitive sites were -26 and -36, and sensitive sites -22 and -31 nt upstream of the transcription start site. The TATAAT and TTCCGG sequences recognized by Xc RNAP were proposed to be the -10 and -35 region, respectively. The spacing between these two regions is 20 bp, which is not well consistent with the E. coli sigma 70 consensus. Taken together, the results showed that the promoter structure recognized by purified Xc RNAP are similar, but not identical to those recognized by E. coli Es70.
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