Promoter Escape by RNA Polymerase II
DOWNSTREAM PROMOTER DNA IS REQUIRED DURING MULTIPLE STEPS OF
EARLY TRANSCRIPTION*
Xiaoxue
Wang,
Lori
Spangler, and
Arik
Dvir
From the Department of Biological Sciences, Oakland University,
Rochester, Michigan 48309
Recent evidence, obtained in a reconstituted
RNA polymerase II transcription system, indicated that
the promoter escape stage of transcription requires template DNA
located downstream of the elongating polymerase. In the absence of
downstream DNA, very early elongation complexes are unable
to synthesize transcripts longer than ~10-14 nucleotides. In
contrast, once transcripts longer than ~15 nucleotides have
been synthesized, an extended region of downstream DNA is no
longer required (Dvir, A., Tan, S., Conaway, J. W., and Conaway,
R. C. (1997) J. Biol. Chem. 272, 28175-28178).
In this work, we sought to define precisely when, during the synthesis
of the first 10-15 phosphodiester bonds, downstream DNA is required.
We report that, for complete promoter escape, downstream DNA extending
to position 40/42 is required. The polymerase can be forced to arrest
at several points prior to the completion of promoter escape by
removing downstream DNA proximally to positions 40/42. The positions at
which the polymerase arrests appear to be determined by the length of
available downstream DNA, with arrest occurring at a relatively fixed
position of ~28 nucleotides to the distal end of the template. A
similar requirement is observed for transcription initiation,
i.e. the formation of the first phosphodiester bond of
nascent transcripts. In addition, we show that the requirement for a
downstream region is independent of downstream DNA sequence, suggesting
that the requirement reflects a general mechanism. Taken together, our
results indicate (i) that downstream DNA is required continuously
through the synthesis of the first 14-15 phosphodiester bonds
of nascent transcripts, and (ii) that a major conformational change in
the transcription complex likely occurs only after the completion of
promoter escape.
*
This work was supported by Grants MCB-9817004 and
MCB-0215992 from the National Science Foundation and by the Oakland
University Research Excellence Program in Biotechnology.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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
