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 f ootprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are tr eated with elevated levels of ribonucleases A and T1, the nascent transcrip t can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary comp lexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an ac tive ternary complex. However, cleavage by ribonuclease 1 is restricted, an d gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first reg ion extends through the first 15-16 nucleotides from the 3'-OH terminus; th e second region extends from position 30 out to position 45. We interpret t hese regions of partial protection as defining two RNA product binding site s on the RNA polymerase that bind the product to the enzyme during elongati on Our results rule out the existence of a stable RNA-DNA hybrid in these t ernary complexes of greater than 3 base pairs in length.