FACTOR-DEPENDENT RELEASE OF NASCENT RNA BY TERNARY COMPLEXES OF VACCINIA RNA-POLYMERASE

Factor-dependent transcription termination during synthesis of vaccini a early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU si gnal in the nascent transcript. The choice of termination site is flex ible and is determined by a kinetic balance between nascent chain elon gation and the transmission of the RNA signal to the polymerase. To el iminate ongoing elongation as a variable, we have established a system to study transcript release by purified ternary complexes halted at s t defined template position 50-nucleotides 3' of the first U residue o f the termination signal. Release of the nascent RNA depends on the va ccinia termination factor (VTF) and an ATP cofactor. Transcript releas e is blocked by BrUMP substitution within the termination signal of th e nascent RNA, in these respects, the release reaction faithfully mimi cs the properties of the termination event. We demonstrate that ternar y complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU signal is situated 20 nucleotides from the gro wing point of the nascent chain. Ribonuclease footprinting of the arre sted ternary complexes defines a nascent RNA binding site oil the poly merase elongation complex that encompasses a 16-21 nucleotide RNA segm ent extending proximally from the 3' end of the chain. We surmise that access of VTF to the signal sequence is prevented when UUUUUNU is bou nd within the nascent RNA binding site. Hence, physical not kinetic co nstraints determine the minimal distance between the signal and potent ial sites of 3' end formation.