Abstract
During transcription elongation the nascent RNA remains base-paired to the template strand of the DNA before it is displaced and the two strands of the DNA reanneal, resulting in the formation of a transcription "bubble" of approximately 10 bp. To examine how the length of the RNA-DNA hybrid is maintained, we assembled transcription elongation complexes on synthetic nucleic acid scaffolds that mimic the situation in which transcript displacement is compromised and the polymerase synthesizes an extended hybrid. We found that in such complexes bacterial RNA polymerase exhibit an intrinsic endonucleolytic cleavage activity that restores the hybrid to its normal length. Mutations in the region of the RNA polymerase near the site of RNA-DNA separation result in altered RNA displacement and translocation functions and as a consequence in different patterns of proofreading activities. Our data corroborate structural findings concerning the elements involved in the maintenance of the length of the RNA-DNA hybrid and suggest interplay between polymerase translocation, DNA strand separation, and intrinsic endonucleolytic activity.
Highlights
Structural and biochemical studies of multisubunit RNAPs have implicated structurally conserved motifs located at the Grants GM38147
These other structural elements are not directly involved in RNA displacement but rather are thought to prevent reassociation of RNA after it has been displaced from the template DNA and to form an extensive network of interactions resulting in stabilization of the nucleic acid components of the elongation complex (EC) [6, 9, 10]
In this work, using ECs assembled on single-stranded DNA scaffolds with wild type (WT) and mutant RNAPs, we demonstrate that bacterial RNAP can maintain the length of the RNA-DNA hybrid as a result of an intrinsic endonuclease activity
Summary
Purification of RNAP and Polymerase Activity Assay—WT 2-mercaptoethanol). Primer extension was achieved by incuba-. The Ј⌬252–263 mutant, pausing upon incorporation of 5 nt, was able to complete transcription and produced a full-length product that extended to the end of the template with high efficiency This suggests that removal of the lid eliminates the physical barrier for the growing RNA-DNA hybrid that causes instability in EC in full agreement with the previous studies [7, 8]. WT RNAP or the lid deletion mutant (Fig. 5B) These data together with the primer extension experiment described above (Fig. 5A) suggest that the switch 3 element may play an important role in the translocation event because EC8 is in a pretranslocated state and translocation is required for both substrate incorporation (forward translocation) and endonucleolytic activity (backward translocation)
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