Direct versus limited-step reconstitution reveals key features of an RNA hairpin-stabilized paused transcription complex.
We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9-10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.
Kyzer
Scotty
S
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Ha
Kook Sun
KS
Landick
Robert
R
Palangat
Murali
M
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D035602:Transcriptional Elongation Factors
D011756:Diphosphates; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases