Promoter Clearance 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...

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 ...

Structure and function in promoter escape by T7 RNA polymerase.

Promoter clearance by T7 RNA polymerase. Initial bubble collapse and transcript dissociation monitored by base analog fluorescence.

Footprinting, fluorescence, and x-ray structural information from the initial, promoter-bound complex of T7 RNA polymerase describes the very beginning of the initiation of transcription, whereas recent fluorescence and biochemical studies paint a preliminary picture of an elongation complex. The current work focuses on the transition from an initially transcribing, promoter-bound complex to an...

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...