Transcript cleavage by Thermus thermophilus RNA polymerase. Effects of GreA and anti-GreA factors.
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.
Hogan
Brian P
BP
Department of Chemistry, CB #3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Hartsch
Thomas
T
Erie
Dorothy A
DA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002021:Buffers; D012333:RNA, Messenger; D014157:Transcription Factors; C075886:GREA protein, Rickettsia prowazekii; D012321:DNA-Directed RNA Polymerases