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In this study, we applied column chromatography and purified
the RNA polymerase from the wild type strain of Xc.11. To
achieve this purpose, 70.19 g of wet cell were collected and
then broken by sonication, fractionation by 38% to 65% ammonium
sulfate cut . The sample were applied into Heparin-Sepharose
CL-6B column, and collected from bufferA with 0.2M, 0.35M and
0.5M KCl. The samples with enzyme activity eluted from 0.35M
KCl were applied into Bio- Gel A 1.5 column and DEAE-Sephadex A
25 column and obstain 5.31mg purified RNA polymerase holoenzyme.
The samples with enzyme activ- ity eluted from 0.5 M KCl buffer
A were applied into Bio - Rex 70 column and Bio-Gel A 1.5
column and finally obtained 3.47mg holo- enzyme . In order to
check the purity of the purified RNA polymerase,a two
dimentional gel electrophoresis was performed and only four
spots( beta'', beta,sigma and alpha) were found after staining.
In the mean time,the 81.51kDa sigma factor of Xc11 was isolated
from the SDS-PAGE and used to immunize the rabbit. Antiserum
against the σsubunit of Xc.11 was then prepared and the title
was deter- mined by Western-blot analyses. In order to examine
the enzymatic activity of the Xc. 11 RNA polymerase several
DNA templates were used in the transcription reactions in
vitro found that (1) Xc 11 RNA polymerase can recog- nize E.
coli lac promoter and produced a 198 nt transcript . ( 2) The
purified Xc. 11 RNA polymerase can recognize either the E. coli
thr promoter or terminator and produced a 164 nt terminated
transcript and a 300 nt read - through transcript as E. coli
RNA polymerase . (3) The purified Xc11 RNA polymerase also can
recog- nized T7A1 promoter and produce 110 nt RNA product . (4)
The pro- moter of xpsA is recognized well by the purified RNA
polymerase and E. coli RNA polymerase.
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