Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes.
Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells. We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex. However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45. We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation. Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length.
Milan
S
S
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720-3202, USA.
D'Ari
L
L
Chamberlin
M J
MJ
D001483:Base Sequence; D016386:Chromosome Walking; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008956:Models, Chemical; D012329:RNA, Bacterial / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic