Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation

Abstract

Ternary complexes of RNA polymerase II, bearing the nascent RNA transcript, are intermediates in the synthesis of all eukaryotic mRNAs and are implicated as regulatory targets of factors that control RNA chain elongation and termination. Information as to the structure of such complexes is essential in understanding the catalytic and regulatory properties of the RNA polymerase. We have prepared complexes of purified RNA polymerase II halted at defined positions along a DNA template and used RNase footprinting to map interactions of the polymerase with the nascent RNA. Unexpectedly, the transcript is sensitive to cleavage by RNases A and T1 at positions as close as 3 nucleotides from the 3'-terminal growing point. Ternary complexes in which the transcript has been cleaved to give a short fragment can retain that fragment and remain active and able to continue elongation. Since DNA.RNA hybrid structures are completely resistant to cleavage under our reaction conditions, the results suggest that any DNA.RNA hybrid intermediate can extend for no more than 3 base pairs, in dramatic contrast to recent models for transcription elongation. At lower RNase concentrations, the transcript is protected from cleavage out to about 24 nucleotides from the 3' terminus. We interpret this partial protection as due to the presence of an RNA binding site on the polymerase that binds the nascent transcript during elongation, a model proposed earlier by several workers in preference to the hybrid model. The properties of this RNA binding site are likely to play a central role in the process of transcription elongation and termination and in their regulation.