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Molecular and Cellular Biology, May 2001, p. 3166-3178, Vol. 21, No. 9
Istituto Pasteur
Received 10 August 2000/Returned for modification 7 September
2000/Accepted 5 February 2001
Quantitative analysis of multiple-hit potassium permanganate
(KMnO4) footprinting has been carried out in vivo on
Saccharomyces cerevisiae 5S rRNA genes. The results fix the
number of open complexes at steady state in exponentially growing cells
at between 8 and 17% of the 150 to 200 chromosomal copies. UV and
dimethyl sulfate footprinting set the transcription factor TFIIIB
occupancy at 23 to 47%. The comparison between the two values suggests
that RNA polymerase III binding or promoter opening is the
rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA
elongation in vivo by cordycepin confirms this result. An experimental
system that is capable of providing information on the mechanistic
steps involved in regulatory events in S. cerevisiae cells
has been established.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.9.3166-3178.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
RNA Polymerase III Transcription Complexes on
Chromosomal 5S rRNA Genes In Vivo: TFIIIB Occupancy and Promoter
Opening
Fondazione Cenci
Bolognetti, c/o Dipartimento di Genetica e Biologia Molecolare,
Università di Roma, La Sapienza,1
Centro di Studio per gli Acidi Nucleici,
CNR,3 and Osservatorio Astronomico di
Roma,4 Rome, Italy, and Department of
Molecular and Cell Biology, University of California Berkeley,
Berkeley, California2
*
Corresponding author. Mailing address: Centro di Studio
per gli Acidi Nucleici, CNR, Dipartimento di Genetica e Biologia
Molecolare, Universita' di Roma, La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy. Phone: 390649912897. Fax: 390649912500. E-mail:
Rodolfo.Negri{at}uniroma1.it.
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