Phage promoter structure and recognition by T7 and SP6 RNA polymerases

Abstract

In specific protein-DNA interactions, protein recognizes a particular DNA sequence of particular DNA structure. In this research phage RNA polymerase-promoter systems were stucied as examples of protein-DNA interactions. First, we were interested in the structural basis for the region of T7 promoter $\phi$10, which was known to be one of the strongest promoters in vivo and vitro. Just upstream of the gene 10 in bacteriophge T7 genome there exist expression regulatory sequences for phage T7 RNA polymerase transcription and host E. coli ribosome translation. These DNA regions have been examined for sequence-directed DNA bending. Permutation analysis of DNA fragments in these regions reveraled that about 30 base pairs downstream of the translation initiation codon ATG there appears to exist a sequencedirected DNA bending. The bent region does not contain any oligo(A) tract which is known to cause bending of DNA. Calculations of the accumulated wedge angles of DNA fragments based on wedge angles revealed a good correlation between the expermintal gel mobility retardation degrees and calculated DNA axis deflection angles of all the permuted DNA fragments. Theese data support the wedge model of DNA curvature. In order to describe real DNA axis curvature a calculation method of three dimensional tracking of DNA axis path was developed. Also, a computer program was developed to calculate the coordinates of DNA backbone and visualize three dimensional structure of DNA bending by computer graphics. Second, in order to identify the base pairs responsible for the distinction between T7 and SP6 promoters`` specific interactios with their own RNA polymerases, we made multiple base pair substitutions for phage T7 and SP6 consensus promoter sequences. Based upon comparison between the two consensus sequences and point mutation results of the T7 promoter, our target region was chosen to be the base pairs at -12, -10, -9 and -8, upstream of the transcription initiation site +1. ...