Transcript Cleavage by Thermus thermophilus RNA Polymerase: EFFECTS OF GreA AND ANTI-GreA FACTORS

Brian P Hogan,Thomas Hartsch,Dorothy A Erie

doi:10.1074/jbc.m108737200

Brian P Hogan, Thomas Hartsch + Show 1 more

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https://doi.org/10.1074/jbc.m108737200

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Abstract

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity of Thermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus and Thermus aquaticus. The thermostable GreA factors contact the 3'-end of RNA, stimulate the intrinsic cleavage activity of T. thermophilus RNA polymerase, and increase the k(app) of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal "basic patch" region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase.

Highlights

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript
To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity of Thermus thermophilus RNA polymerase
To further ensure that no transacting transcript cleavage factor copurified with T. thermophilus RNA polymerase (RNAP), 10 ␮g of RNAP were run on SDS-PAGE and visualized by silver staining

Summary

Introduction

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. It has been suggested that the same amino acids that are responsible for nucleotide incorporation may be responsible for this cleavage activity [10, 11] If this hypothesis is true, it would indicate large scale movements of the catalytic site relative to the RNA transcript and the DNA template [12,13,14]. Such conformational changes have been investigated in vitro using stalled complexes formed by NTP deprivation or physical obstruction [15,16,17].

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