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Molecular and Cellular Biology, May 2001, p. 3166-3178, Vol. 21, No. 9
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

G. Costanzo,1 S. Camier,2 P. Carlucci,3 L. Burderi,4 and R. Negri3,*

Istituto Pasteur---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

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.

* 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.

Molecular and Cellular Biology, May 2001, p. 3166-3178, Vol. 21, No. 9
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.9.3166-3178.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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