lac Promoter mutation Pr115 generates a new transcription initiation point.

The Eecherichiu coli luc P'115 mutation changes the base-pair where in vitro RNA synthesis initiates from an A/T to a T/A (Maquat et al., 1980). Such an alt,eration results in a catabolite gene activator protein-independent phenotype (Reznikoff, 1976; Maquat et al., 1980). In the absence of catabolite gene activator protein and CAMP, the altered base-pair of the mutant DNA is not used in vitro to initiate messenger RNA, but RNA synthesis is initiated at a new site estimated to be 12 base-pairs downstream (+ 13) from the P + transcription initiation site (+ 1). In the presence of the positive affecters, lacPrl15-directed transcription initiates near the + 1 site. Thus, CAMP-activated catabolite gene activator protein at least in part overcomes the effect that the P'llS base-pair alteration has on the stitrt site of RNA synthesis. The ZacPrl 15 studies suggest that catabolite gene act&&or protein stimulates transcription initiation only at discrete locstions (at) or near + I but, not at or near + 13). Pr (class III) luc promoter mutations are sequence changes which enhance the expression of the lac operon in the absence of the positive aficctors: the catabolite gene activator protein and adenosine 3', 5'-cyclic phosphate. The mutant promoters are tfhought to have an increased affinity for RNA polymerase relative to the wild-type promoter such that the binary RNA polymerase-promoter complex forms at, a faster rate and transcription initiation occurs more frequently (Reznikoff, 1976; Maquat & Reznikoff, 1978). This has been found to be the case for ail P' mutations analyzed to date. Sequence analysis has indicated that the lacP'115 mutation is an A/T + T/A transversion at the +l site (Maquat et al.: 1980; see Fig. l), t,he major start site of ilz vitro Zac messenger RNA synthesis (Maizels, 1973; Majors, 1975; Maquat & Rezni-koff. 1978). It is of interest t,o determine how an alteration of the base-pair which codes for the first mRNA nucleotide results in a CAP?-CAMP-independent phenotype. We describe experiments utilizing an in vitro t,ranscriptional system which analyzes the funct,ional interaction between RNA polymerase and ZucPp115 DNA. To approximat,r t'he start site of in vitro ZucP'115 RNA synthesis, 2acPrl15-containing restriction fragments were used as templates in the transcriptional assay. The mobility of the major transcripts produced by Hue111 203 kzcP+ corresponds to RNA molecules which are 63 and 64 bases (Maquat & Reznikoff, 1978; Maquat et al.. 1980; see Fig. 2). This is true …