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Molecular and Cellular Biology, April 2001, p. 2641-2649, Vol. 21, No. 8
Division of Gene Regulation and Expression,
Wellcome Trust Biocentre, School of Life Sciences, University of
Dundee, Dundee DD1 5EH, Scotland, United Kingdom
Received 20 December 2000/Accepted 22 January 2001
The assembly, disassembly, and functional properties of
transcription preinitiation complexes (PICs) of human RNA polymerase I
(Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that
these complexes contained the factors required to support initiation of
transcription, SL1, upstream binding factor (UBF), and Pol I. We have
demonstrated that, throughout a single round of transcription, SL1 and
UBF remain promoter bound. Moreover, the promoter-bound SL1 and UBF
retain the ability to function in transcription initiation. SL1 has a
central role in the stable association of the PIC with the promoter
DNA. The polymerase component of the PIC is released from the promoter
during transcription yet is efficiently recycled and able to reinitiate
from "poised" promoters carrying SL1 and UBF, since the PICs
captured on the immobilized templates sustained multiple rounds of
transcription. Kinetic analyses of initiation of transcription by Pol I
revealed that Pol I-dependent transcription is rate limited in a step
subsequent to recruitment and assembly of Pol I PICs. The rate of RNA
synthesis is primarily determined by the rates at which the polymerase
initiates transcription and escapes the promoter, referred to as
promoter clearance. This rate-limiting step in Pol I transcription is
likely to be a major target in the regulation of rRNA gene expression.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2641-2649.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
A Step Subsequent to Preinitiation Complex Assembly
at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA
Polymerase I-Dependent Transcription
*
Corresponding author. Mailing address: Division of Gene
Regulation and Expression, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Phone: 44-1382-344242. Fax: 44-1382-348072. E-mail:
j.zomerdijk{at}dundee.ac.uk.
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