Quantitative analysis of multiple-hit potassium permanganate (KMnO 4 ) 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.Yeast RNA polymerase III transcription machinery components and their interaction with promoter elements are known in detail (for reviews, see references 19, 22, and 45). 5S rRNA transcription requires TFIIIB, which is the initiation factor proper of RNA polymerase III, and two assembly factors, TFIIIA and TFIIIC (25). The in vitro topography of transcription factor complexes is well described thanks to extensive DNA footprinting studies (8,25,26), protein-DNA cross-linking (2, 9, 27), and protein subunit assembly studies (13,29). Analysis of KMnO 4 sensitivity on open complexes and stalled elongation complexes has suggested structural analogies to bacterial polymerase mechanisms (25,28). In spite of all the available information on the in vitro systems, very little is known about the organization of transcription complexes on 5S genes inside yeast cells. A weak modulation of DNase I cutting has recently been observed in the 5Ј-flanking sequence of 5S genes on episomes in vivo (31, 32). Previous attempts to reveal transcription factor-DNA interactions on the 5S chromosomal genes in vivo did not provide conclusive information. These difficulties are primarily due to (i) the low occupancy by transcription complexes of the multiple copies of the genes present in the cell (14), (ii) the lack of studies making use of noninvasive footprinting techniques, and (iii) the lack of proper nontranscribing control conditions allowing clear distinctions to be made between changes due to transcription complexes and those due to chromatin organization. In this study we overcome these problems. UV (3, 5), dimethyl sulfate (DMS) (21), and KMnO 4 (37) footprinting can be performed directly on growing cells. This approach involves short exposure times with minimal impact on the biological structure to be investigated. Histone-DNA interactions have only a small influence on UV irradiation and DMS reactivity patterns, allowing selective visualization of transcription factor footprints (4, 21). KMnO 4 is a highly specific probe for open-complex formation. A system was developed to transcribe 5S rRNA genes in the absence of TFIIIA (12). In this system, yeast cells devoid of TFIIIA, which therefore cannot form transcription complexes on endogenous chromo...