Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA.

S Shuman,B Moss

doi:10.1016/s0021-9258(18)68775-7

Abstract

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.

Highlights

Transcription terminationin vitroby vaccinia RNA Birnstiel et al, 1985).This owes, in large part, to thefact that polymeraseis dependent on a trans-acting factoVr,TF, most eukaryotic RNAs derive their 3‘ termini via processing that is associatedwith, if not identicalto, the vaccinia events rather than as a consequence of transcription termimRNA capping enzyme
We have focused on vaccinia virus early transcription as a model system for studying termination of mRNA synthesis
Factor-independent termination in Escherichia coli typically occurs within a T run downstream of a G:C-rich dyad symmetry element (Rosenberg and Court, 1979).In uitro, the substitution of analogs that stabilize G:C base pairs (e.g. BrCTP, ICTP) stimulates termination,while substitution of analogs that weaken G:C pairing (e.g. ITP) promotes read through

Summary

THEJOURNALOF BIOLOGICACHLEMISTRY

Vol 263, No 13,Issue of May 5, pp. 6220-6225,1988 Printed in U S A. An in uitro transcription system specific for vaccinia early genes has been exploited to define a cis-acting sequence TTTTTNT in thenontranscribed template strandthat is required for termination (Rohrmann et al, 1986). This seven nucleotide sequence is experiments, unless specified otherwise. Since termination occurs at sitesdistal to the signal, the cognate sequence UUUUUNU is expressed in the RNA transcript We have approached this issue by studying the effects of ribonucleotide base analogs on the efficiency of VTF-dependent termination in uitro.

EXPERIMENTAL PROCEDURES

Effect ofN T P analogs on vaccinia RNA polymerase

RNA Signal for Transcription Termination

DISCUSSION

Nuclease sensitivity of RNA products

Percent solubilized cFJm