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Vol. 13, No. 16, pp. 2134-2147, August 15, 1999
subunit
1 Department of Bacteriology, University of Wisconsin,
Madison, Wisconsin 53706 USA; 2 Howard Hughes Medical
Institute, Waksman Institute and Department of Chemistry, Rutgers
University, Piscataway, New Jersey 08854 USA
We demonstrate here that the previously described bacterial promoter
upstream element (UP element) consists of two distinct subsites, each
of which, by itself, can bind the RNA polymerase holoenzyme 
subunit
carboxy-terminal domain (RNAP CTD) and stimulate transcription.
Using binding-site-selection experiments, we identify the consensus
sequence for each subsite. The selected proximal subsites (positions
46 to 38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions 57 to 47; consensus 5'-AWWWWWTTTTT-3')
stimulate transcription up to 16-fold. RNAP has subunit composition
2
'
and thus contains two copies of CTD.
Experiments with RNAP derivatives containing only one copy of CTD
indicate, in contrast to a previous report, that the two CTDs
function interchangeably with respect to UP element recognition.
Furthermore, function of the consensus proximal subsite requires only
one copy of CTD, whereas function of the consensus distal subsite
requires both copies of CTD. We propose that each subsite
constitutes a binding site for a copy of CTD, and that binding of an
CTD to the proximal subsite region (through specific interactions
with a consensus proximal subsite or through nonspecific interactions
with a nonconsensus proximal subsite) is a prerequisite for binding of
the other CTD to the distal subsite.
[Key Words:
Promoter; RNA polymerase; subunit; UP
element; transcription initiation]
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