Abstract
Effects of mutations around the phage SP6 transcription initiation site on SP6 RNA polymerase's selection of initiation site were studied. In the in vitro transcription reactions, the limiting concentration of a ribonucleotide causes the SP6 RNA polymerase to stall long enough only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result, a series of RNA oligomers comprises a sequencing ladder, and abortive initiation cycling products up to 6-mer are made in high yield. Precise sizing of the product RNAs from the elongation pausings determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, transcription still starts only at the +1 site. But, the mutant containing TATCC from -4 to +1 C. We propose that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. It is also suggested that the sequence-dependent perturbations of DNA helical structure, for example D to B form, may shift the initiation site.