Functional topography of nascent RNA in elongation intermediates of RNA polymerase

To determine the dynamics of transcript extrusion from Escherichia coli RNA polymerase (RNAP), we used degradation of the RNA by RNases T1 and A in a series of consecutive elongation complexes (ECs). In intact ECs, even extre mely high doses of the RNases were unable to cut the RNA closer than 14-16 nt from the 3' end. Our results prove that ail of the cuts detected within the 14-nt zone are derived from the EC that is denatured during inactivatio n of the RNases. The protected zone monotonously translocates along the RNA after addition of new nucleotides to the transcript. The upstream region o f the RNA heading toward the 5' end is cleaved and dissociated from the EC, with no effect on the stability and activity of the EC. Most of the curren t data suggest that an 8- to 10-nt RNA DNA hybrid is formed in the EC. Here , we show that an 8- to 10-nt RNA obtained by truncating the RNase-generate d products further with either GreB or pyrophosphate is sufficient for the high stability and activity of the EC. This result suggests that the transc ript-RNAP interaction that is required for holding the EC together can be l imited to the RNA region involved in the 8- to 10-nt RNA.DNA hybrid.