Research of gene promoter in Mycobacterium tuberculosis using Fixed-RNA polymerase holoenzymes with the developed resin.

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Recombinant RNA polymarase (RNAP) holoenzymes containing different sigma factors, SigA, SigB, SigC, SigD, SigE, SigG, SigH, SigI, respectively, in Mycobacterluin tuberculosis were purified using Escherichia coil expression system. We prepared eight kinds of fixed RNAP holoenzymes with the developed resin. Also, genome library was constructed with PCR using four primers designed from the appearance frequency in M. tuberculosis genome. From this genome library, we selected the DNA fragments, which have high affinity for the fixed RNAP holoenzymes and were diffused with transcription for one cycle.As a result, SigA type promoters had the consensus sequence TTGACA<16/l7bp>TATAAT as same as E. coil sigma7O promoter consensus sequence. SigH type promoters were conserved 35 regions, comparing with ECF type promoter consensus sequence CCGGAACTT<16/17bp>TCTgA. However, SigE type promoters had no similarity with ECF type promoter, the consensus sequence was predicted GG(aa/gc) (C/A)K18bp>(c/g)GTT(g/c).