Groups on the Outside of the DNA Helix Affect Promoter Utilization by T7 RNA Polymerase

Abstract

INTRODUCTION
One of the important features of RNA polymerase is its ability to recognize promoter regions in DNA at which the enzyme initiates RNA synthesis. Since RNA polymerase probably recognizes promoter regions while the DNA is in the closed double-helical form (Chamberlin 1974), how are the specific nucleotide sequences that govern this interaction recognized by RNA polymerase with the bases hydrogen bonded to each other in the interior of the DNA double helix? RNA polymerase might “read” the promoter base sequence by interacting with specific residues in either the major or minor groove of the DNA double helix. This suggests that promoter recognition would depend on residues accessible on the outside of the duplex that are not necessarily involved in the specificity of base-pairing. We have investigated this scheme of promoter recognition by replacing certain bases in promoter regions of DNA with analogs that form the normal Watson-Crick base-pairing interactions but are altered in residues that appear in either the major or minor groove of the DNA double helix. Alterations in the ability of T7 RNA polymerase to utilize these modified promoter regions may suggest which groups on the nucleotide bases are important in the promoter recognition process.

Our experimental scheme is presented in Figure 1. We have employed a phage hybrid, hy8, a recombinant phage containing segments of both of the genomes of the related phages T3 and T7 (Golomb and Chamberlin 1974a). Hy8 has two T7 RNA polymerase promoters at map positions of 83% and 97% on the...