Abstract ▼ AI-Helper

A library of lacUV5 promoter mutants carrying the base substitutions at and around the +1 site has been generated by polymerase chain reaction (PCR) using mismatched primer, which contains a mixture of G, A, T, and C in the ratio of 2:2:1:1 at the position corresponding to +1 site. The mutants in wh...

A library of lacUV5 promoter mutants carrying the base substitutions at and around the +1 site has been generated by polymerase chain reaction (PCR) using mismatched primer, which contains a mixture of G, A, T, and C in the ratio of 2:2:1:1 at the position corresponding to +1 site. The mutants in which +1 A was changed to T or C and wild-type were obtained. But the change of A to G has not been found yet. Also unexpected mutations which are presumed to be introduced during the amplification, occurred at -18 position where G was deleted or changed to a and -38 position where G was changed to A. The change of G to T was also found at wild-type opsition +8. The transcription initiation sites of all the lacUV5 promoter variants were determined by sizing the run-off transcripts. The predominat transcriptional startpoint appears to shift to the wild-type position +2 when the wild-type position +1 A is changed to T or C. Base substitution at -38 position and the deletion of G at -18 position do not seem to affect the selection of transcription initiation site. Now, it is thought that E. coli RNA polymerase prefers a purine as a initiating nucleotide, so initiates at the next purine when a purin of +1 site is changed to a pyrimidine.

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