Start site selection at lacUV5 promoter affected by the sequence context around the initiation sites

The effects of single base pair substitutions at the initiation sites of lacUV5 promoter on the transcription start site selection by E. coli RNA polymerase were systematically studied. Transcription start sites were mapped by sizing the cytosine-specifically terminated transcripts produced in vitro by using a chain terminator 3'-deoxycytidine 5'-triphosphate (3'-dCTP) in transcription reactions. Transcription of a prototype lacUV5 promoter initiated with three purines (-1G, +1A and +2A; +1 representing the predominant start site) located 6-8 bp downstream from the Pribnow box. All the substitutions affected the start site selection, resulting in a change in the number of start sites (from 3 to 2 or 1) and/or a shift of the major start site (to -1 or +2). None of the variants started outside the 3-bp region and at the positions substituted by a pyrimidine. Purine-to-pyrimidine changes suppressed not only initiation at the substituted position but also, in some cases, at the other purine position. Purine-to-purine changes also shifted the major start site or suppressed the initiation at other sites. Changes at -2 and +5 also affected the start site selection. Thus, the sequence context around the initiation sites of lacUV5 promoter strongly influences the selection of initiating nucleotides by E. coli RNA polymerase.