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T7 promoter release mediated by DNA scrunching.

ABSTRACT: Transcription initiation includes a phase in which short transcripts dissociate from the transcription complex and the polymerase appears not to move away from the promoter. During this process DNA may scrunch within the complex or the polymerase may transiently break promoter contacts to transcribe downstream DNA. Promoter release allowing extended downstream movement of the polymerase may be caused by RNA-mediated disruption of promoter contacts, or by limits on the amount of DNA that can be scrunched. Using exonuclease and KMnO4 footprinting of T7RNAP transcription complexes we show that the DNA scrunches during progression through initial transcription. To determine whether promoter release is determined by RNA length or by the amount of DNA scrunched, we compared release at promoters where the polymerase is forced to initiate at +2 with those where it initiates at +1. For RNAs of identical length, release is greater when more DNA is scrunched. Release is inhibited when a nick introduced into the template relieves the strain of scrunching. DNA scrunching therefore makes an important contribution to T7 promoter release.

SUBMITTER: Brieba LG

PROVIDER: S-EPMC125763 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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T7 promoter release mediated by DNA scrunching.

Brieba L G LG Sousa R R

The EMBO journal 20011201 23

Transcription initiation includes a phase in which short transcripts dissociate from the transcription complex and the polymerase appears not to move away from the promoter. During this process DNA may scrunch within the complex or the polymerase may transiently break promoter contacts to transcribe downstream DNA. Promoter release allowing extended downstream movement of the polymerase may be caused by RNA-mediated disruption of promoter contacts, or by limits on the amount of DNA that can be s ...[more]

PMID: 11726518

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Project description:Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4-7 nt) while marginally increasing long abortives (9-11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K(d) by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1-2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

Dataset Information

T7 promoter release mediated by DNA scrunching.

Publications

T7 promoter release mediated by DNA scrunching.

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