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The IacUV5 promoter mutants carrying base substitutions at +1 site were generated by polymerase chain reaction (PCR) using mismatched primers. The transcription initiation sites of lacUV5 wild-type promoter and its initiation site variants were determined by sizing both run-off transcripts produced from linearized templates, and nucleotide-specific pausing product RNAs produced by using 3´-deoxycytidine 5-triphosphate instead of cytidine 5´-triphosphate in in vitro transcription reactions. From 1ac1UV5 wild-type promoter, transcription initiates also at 1 G and +2 A sites, while the major initiation site is +1 A. When the wild-type A at + 1 site was changed to a pyrimidine T or C, however, transcriptions initiated only at +2 A. Also unexpected mutations occurred at -38, -18, and +8 positions, which are presumed to be introduced during the PCR amplification. The base substitutions at -38, -18 and +8 positions, and a deletion at -18 position did not affect the selection of transcription initiation site. Thus, E. coli RNA polymerase appears to prefer a pyrimidine-girt purine located at a flexible distance from the -10 region of promoter as an initiating nucleotide, and so initiates only at +2 A when A at +1 site is changed to a pyrimidine.
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