Promoter and nonspecific DNA binding by the T7 RNA polymerase

Evidence for DNA bending at the T7 RNA polymerase promoter.

Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A com...

Structure and function in promoter escape by T7 RNA polymerase.

RNA-binding site in T7 RNA polymerase.

Recent models of RNA polymerase transcription complexes have invoked the idea that enzyme-nascent RNA contacts contribute to the stability of the complexes. Although much progress on this topic has been made with the multisubunit Escherichia coli RNA polymerase, there is a paucity of information regarding the structure of single-subunit phage RNA polymerase transcription complexes. Here, we pho...

Promoter specificity determinants of T7 RNA polymerase.

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage ...

Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase.

Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix...