Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase.

C Andrews,J P Richardson

doi:10.1016/s0021-9258(18)89096-2

Abstract

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.

Highlights

390 to 470 Nucleotides Downstream from the A, PromoterT 7 Dl11 DNAwas chosen for studies of the effects of rho on dissociation because it has a single strong promoter that is used almost exclusively when the molar ratio of RNA polymerase to DNA is less than one [16]
To determine where rho acts to terminate transcripts initiatedat the A, promoter, we analyzed the size distribution of the RNA products made from transcription of T 7 Dl11 DNA in the presence and absence of rhofactor.Theresults show thatmanydifferent RNA molecules with sizes ranging from -390 nucleotides to >2000 nucleotides were made in the presence of rho that were not made in its absenc(eFig. 1, compare lane A with lane F). a small amount of an RNA with a length of 224 nucleotides was made in the presence and absence of rho, presumably from initiation at theA. promoter and termination at the end of the DNA [16, 17]
To determine whether the points of termination in the region from 390 to 470 nucleotides downstream from the A1promoter correspond to pause sites within that region as shownpreviouslyfor AtRl [21, 22], the rhoterminated RNA was compared with total RNA from isolated transcription complexes prepared by synthesis for 40 s without rho and isolatebdy centrifugation througha small column of Sephadex G-50, a procedurethat removes all thenucleoside triphosphates witha quantitative yield of complexes and RNA molecules with morethan 100nucleotides

Summary

Introduction

The [32P]T7 RNAwas prepared by transcription of T 7 DNA (wild type)with E. coli RNA polymerase in the absence of rho factor. To determine whether the points of termination in the region from 390 to 470 nucleotides downstream from the A1promoter correspond to pause sites within that region as shownpreviouslyfor AtRl [21, 22], the rhoterminated RNA was compared with total RNA from isolated transcription complexes prepared by synthesis for 40 s without rho and isolatebdy centrifugation througha small column of Sephadex G-50, a procedurethat removes all thenucleoside triphosphates witha quantitative yield of complexes and RNA molecules with morethan 100nucleotides.

Results

Conclusion