T7 RNA polymerase mutants with altered promoter specificities.

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 ...

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.

A mutation in T7 RNA polymerase that facilitates promoter clearance.

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target...

Substitution of ribonucleotides in the T7 RNA polymerase promoter element.

A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the nontemplate and template...