Abstract
Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance between nascent chain elongation and the transmission of the RNA signal to the polymerase. To eliminate ongoing elongation as a variable, we have established a system to study transcript release by purified ternary complexes halted at a defined template position 50-nucleotides 3' of the first U residue of the termination signal. Release of the nascent RNA depends on the vaccinia termination factor (VTF) and an ATP cofactor. Transcript release is blocked by BrUMP substitution within the termination signal of the nascent RNA. In these respects, the release reaction faithfully mimics the properties of the termination event. We demonstrate that ternary complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU signal is situated 20 nucleotides from the growing point of the nascent chain. Ribonuclease footprinting of the arrested ternary complexes defines a nascent RNA binding site on the polymerase elongation complex that encompasses a 16-21 nucleotide RNA segment extending proximally from the 3' end of the chain. We surmise that access of VTF to the signal sequence is prevented when UUUUUNU is bound within the nascent RNA binding site. Hence, physical not kinetic constraints determine the minimal distance between the signal and potential sites of 3' end formation.
Highlights
The principal findings are: (i) transcript release depends on an accessible UUUUUNU signal in the nascent RNA, (ii) the signal is inaccessible when it is contained within the nascent RNA binding site on the polymerase elongation complex, (iii) bromouridine base substitution within the signal interferes with factor-dependent release, and (iv) release depends on an ATP cofactor
The aforementioned properties and requirements for transcript release by halted polymerase complexes are consistent with all that is known about the composite termination reaction occurring during vaccinia early mRNA synthesis
We provide evidence that a physical constraint to termination sitechoice is imposed by the need to extrude the UUUUUNU signal out of the RNA binding site on the polymerase elongation complex
Summary
Immobilized DNA Templates—The pBS-based G21 plasmid containing a vaccinia early promoter fused to a G-less cassette has been described [18]. The biotinylated DNAs were digested with PvuII, and the restriction fragment containing the transcription cassette was isolated by preparative agarose gel electrophoresis. Transcription activity during purification was assayed as described elsewhere [24] in reactions programmed by a SmaI-cut pSB24 template, which contains a vaccinia early promoter fused to a 382-nucleotide G-less cassette [24]. Transcription in Vitro—Ternary transcription complexes were formed in standard reaction mixtures containing (per 20-l reaction volume) 20 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 2 mM dithiothreitol, 1 mM ATP, 0.1 mM UTP, 1 M [␣-32P]CTP (1000 Ci/mmol), 0.1 mM 3ЈOMeGTP, bead-linked DNA (ϳ100 fmol), and vaccinia RNA polymerase. Elongation reactions were performed as specified in the figure legends
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