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J Biol Chem, Vol. 275, Issue 2, 1485-1494, January 14, 2000
From the Department of Molecular Genetics and Microbiology,
University of Florida, Gainesville, Florida 32610-0266
Prior phenotypic analysis of a vaccinia virus
gene A18R mutant, Cts23, showed the synthesis of longer
than wild type (Wt) length viral transcripts during the intermediate
stage of infection, indicating that the A18R protein may act as a
negative transcription elongation factor. The purpose of the work
described here was to determine a biochemical activity for the A18R
protein. Pulse-labeled transcription complexes established from
intermediate virus promoters on bead-bound DNA templates were assayed
for transcript release during an elongation step that contained
nucleotides and various proteins. Pulse-labeled transcription complexes
elongated in the presence of only nucleotides were unable to release
nascent RNA. The addition of Wt extract during the elongation phase
resulted in release of the nascent transcript, indicating that
additional factors present in the Wt extract are capable of inducing
transcript release. Extract from Cts23 or mock-infected cells was
unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By
itself, purified polyhistidine-tagged A18R protein (His-A18R) was
unable to induce release; however, release did occur in the presence of
purified His-A18R protein plus extract from either Cts23 or
mock-infected cells. These data taken together indicate that A18R is
necessary but not sufficient for release of nascent transcripts. We
have also demonstrated that the combination of A18R protein and mock
extract induces transcript release in an ATP-dependent
manner, consistent with the fact that the A18R protein is an
ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter
but is observed using pulse-labeled transcription complexes initiated
from all three viral gene class promoters. Therefore, we conclude that
A18R and an as yet unidentified cellular factor(s) are required for the
in vitro release of nascent RNA from a vaccinia virus
transcription elongation complex.
Vaccinia Virus Gene A18R DNA Helicase Is a Transcript
Release Factor*
*
This work was supported by National Institutes of Health
Grant AI18094 (to R. C. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 352-392-3128;
Fax: 352-392-3133; E-mail: condit@mgm.ufl.edu.
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