RNA polymerase III transcription complexes on chromosomal 5S rRNA genes invivo: TFIIIB occupancy and promoter opening

Quantitative analysis of multiple-hit potassium permanganate (KMnO4) footpr inting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA gen es. The results fix the number of open complexes at steady state in exponen tially growing cells at between 8 and 17% of the 150 to 200 chromosomal cop ies. UV and dimethyl sulfate footprinting set the transcription factor TFII IB occupancy at 23 to 47%. The comparison between the two values suggests t hat RNA polymerase III binding or promoter opening is the rate-limiting ste p 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 even ts in S. cerevisiae cells has been established.