GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.
The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.
Feng
G H
GH
Department of Biology, Washington University, St. Louis, Missouri 63130.
Lee
D N
DN
Wang
D
D
Chan
C L
CL
Landick
R
R
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D011756:Diphosphates; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006157:Guanosine Monophosphate / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011756:Diphosphates; D012333:RNA, Messenger; D014157:Transcription Factors; C075886:GREA protein, Rickettsia prowazekii; D006157:Guanosine Monophosphate; D012321:DNA-Directed RNA Polymerases