Dataset Information

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.

ABSTRACT: Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

SUBMITTER: Revyakin A

PROVIDER: S-EPMC2754787 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.

Revyakin Andrey A Liu Chenyu C Ebright Richard H RH Strick Terence R TR

Science (New York, N.Y.) 20061101 5802

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one ...[more]

PMID: 17110577

Similar Datasets

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

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.

Publications

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets