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Nucleic Acids Research, 2001, Vol. 29, No. 10 2003-2011
© 2001 Oxford University Press

Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements

Dewang Zhou and Susan M. Lobo-Ruppert*

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 844 BBRB, 845 19th Street South, Birmingham, AL 35294, USA

As compared to the metazoan small nuclear RNAs (snRNAs), relatively little is known about snRNA synthesis in unicellular organisms. We have analyzed the transcription of the Schizosaccharomyces pombe U2 snRNA gene in vivo and in the homologous in vitro system. Deletion and linker-scanning analyses show that the S.pombe U2 promoter contains at least two elements: the spUSE centered at –55, which functions as an activator, and a TATA box at –26, which is essential for basal transcription. These data point to a similar architecture among S.pombe, plant and invertebrate snRNA promoters. Factors recognizing the spUSE can be detected in whole cell extracts by DNase I footprinting and competition studies show that the binding of these factors correlates with transcriptional activity. Electrophoretic mobility shift assays and gel-filtration chromatography revealed a native molecular mass of ~200 kDa for the spUSE binding activity. Two polypeptides of molecular masses 25 and 65 kDa were purified by virtue of their ability to specifically bind the spUSE.

* To whom correspondence should be addressed. Tel: +1 205 975 8159; Fax: +1 205 975 7928; Email: sruppert{at}bmg125.cmc.uab.edu


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