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J Biol Chem, Vol. 274, Issue 36, 25285-25290, September 3, 1999
54 Region I in Trans
and Implications for Transcription Activation
From the Department of Biology, Imperial College of Science
Technology and Medicine, Sir Alexander Fleming Building, Imperial
College Road, London SW7 2AZ, United Kingdom
Control of transcription frequently involves the
direct interaction of activators with RNA polymerase. In bacteria, the
formation of stable open promoter complexes by the
54 RNA polymerase is critically dependent on
54 amino Region I sequences. Their presence correlates
with activator dependence, and removal allows the holoenzyme to engage
productively with melted DNA independently of the activator. Using
purified Region I sequences and holoenzymes containing full-length or
Region I-deleted
54, we have explored the involvement of
Region I in transcription activation. Results show that Region I
in trans inhibits a reversible conformational change in the
holoenzyme believed to be polymerase isomerization. Evidence is
presented indicating that the holoenzyme (and not the promoter DNA
per se) is one interacting target used by Region I in
preventing polymerase isomerization. Activator overcomes this
inhibition in a reaction requiring nucleotide hydrolysis. Region I
in trans is able to inhibit activated transcription by the
holoenzyme containing full-length
54. Inhibition
appeared to be noncompetitive with respect to the activator, suggesting
that a direct activator interaction occurs with parts of the holoenzyme
outside Region I. Stabilization of isomerized holoenzyme bound to
melted DNA by Region I in trans occurs largely
independently of the initiating nucleotide, suggesting a role for
Region I in maintaining the open complex.
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