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. col i RNA polymerase were systematically studied. Transcription start site s were mapped by sizing the cytosine-specifically terminated transcrip ts produced in vitro by using a chain terminator 3'-deoxycytidine 5'-t riphosphate (3'-dCTP) in transcription reactions. Transcription of a p rototype lacUV5 promoter initiated with three purines (-1G, + 1A and 2A; + 1 representing the predominant start site) located 6-8 bp downs tream from the Pribnow box. Alt the substitutions affected the start s ite selection, resulting in a change in the number of start sites (fro m 3 to 2 or 1) and/or a shift of the major start site (to -1 or +2). N one of the variants started outside the 3-bp region and at the positio ns substituted by a pyrimidine. Purine-to-pyrimidine changes suppresse d not only initiation at the substituted position but also, in some ca ses, at the other purine position. Purine-to-purine changes also shift ed the major start site or suppressed the initiation at other sites. C hanges at -2 and +5 also affected the start site selection. Thus, the sequence context around the initiation sites of lacUV5 promoter strong ly influences the selection of initiating nucleotides by E. coli RNA p olymerase.