title: Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase.
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.
authors.affiliation: Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul.
authors.forename: S C
authors.initials: SC
authors.lastname: Nam
authors.forename: C W
authors.initials: CW
authors.lastname: Kang
mesh_terms: D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D014158:Transcription, Genetic
chemical_list: D012321:DNA-Directed RNA Polymerases