Structure-function relationship of E. coli promoter and phage SP6 RNA polymerase

Abstract

Selection of transcription start sites by E. coli RNA polymerase were studied by group mutation of promoter elements and functional amino acid residues of phage SP6 RNA polymerase were mapped by random mutagenesis of polymerase gene. The first, the effects of single base pair substitutions within the initiation region (nucleotide positions -1, +1 and +2) and the -35 region (nucleotide positions from -36 to -31) of the lacUV5 promoter on transcription initiation site selection by E. coli were systematically studied. Transcription start sites were mapped by sizing cytidine specifically terminated transcripts produced by using 3``-deoxycytidine 5``-triphosphate instead of cytidine 5``-triphosphate in in vitro transcription reaction. Transcription of the lacUV5 promoter initiated with three purines (-1G, +1A and +2A; +1 represents a predominant start site) 6-8 nucleotides downstream from the -10 region. When any of these start sites is changed to a pyrimidine, that nucleotide was not selected any more as an initiating nucleotide. Changes of a purine to a pyrimidine at -1 site and A to T at +1 site abolish initiation at another purine site, rendering unique initiations at +1A and +2A, respectively. However, when +1 is a pyrimidine, additional base substitution (G→T) at -2 or +5 position causes transcription to initiate at -1G as well as +2A. Transitions of A to G at +1 and +2 sites excluded the initiation at intact two purine sites and at -1G, respectively. When all of the three starting nucleotides are A, the initiation site could not be precisely determined, perhaps because the initial nascent RNA chain slips to the 5`` direction. The start sites of the lacUV5 promoter variants carrying a single base substitution within the -35 region and an insertion of C at +5 position were determined by the same method. The primary transcription start sites of these variants shifted from +1A to -1G. However, these shifts are not due to the mutations within the -35 region but t...