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Nucleic Acids Research, 1984, Vol. 12, No. 3 1377-1390
© 1984

MOLECULAR BIOLOGY

The in vivo and in vitro initiation site for transcription of the rRNA operon of Saccharomyces carlsbergensis

Jacobus Klootwijk, Martin Ph. Verbeet1, Geertruida M. Veldman2, Victoria C.H.F. de Regt, Harm van Heerikhuizen, Jan Bogerd and Rudi J. Planta

Biochemisch Laboratorium, Vrije Universiteit de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

Received December 1, 1983. Accepted January 3, 1984.

We have performed a detailed analyais of the transcription initiation of the rRNA operon in the yeast Saccharomyces carlsbergensis. Electron microscopic analysis of R-looped pre-rRNA molecules together with a very sensitive S1-nuclease mapping showed the use of only a single transcription start at about 700 bp upstream of the 17S rRNA gene and not of the minor start sites proposed for the very closely related species S. cerevisiae by others [Bayev et al. (5), Swanson and Holland (6)]. The sequence of 730 bp of the initiating region is presented. In vitro transcription in concentrated lysates of yeast spheroplasts in the presence of ({gamma}-SH)ATP or ({gamma}-SH)GTP, followed by purification of the in vitro initiated RNA via Hg-agarose, revealed that on the endogenous template exactly the same site is used for transcription initiation as in vivo.

1Present address: Biochemisch Laboratorium, Rijksuniversiteit Leiden, Wassenaarseweg 64, 2300 RA Leiden, The Netherlands

2Present address: Genetics Institute, 225 Longwood Avenue, Boston, MA 02115, USA


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