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Mol Cell Biol, March 1998, p. 1570-1579, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification and Topological Arrangement of Drosophila Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

Yan Wang and William E. Stumph*

Department of Chemistry and Molecular Biology Institute, San Diego State University, San Diego, California 92182-1030

Received 12 September 1997/Returned for modification 9 October 1997/Accepted 2 December 1997

Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position -40 relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity of Drosophila snRNA genes is intrinsically encoded in the PSE sequence itself. We have investigated the differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs. By using a site specific protein-DNA photo-cross-linking assay, we identified three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa that are in close proximity to the DNA and two additional putative polypeptides of 230 and 52 kDa that may be integral to the complex. The 95-kDa subunit cross-linked at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-linked only to the 3' half of the PSE. The same polypeptides cross-linked to both the U1 and U6 PSE sequences. However, there were significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding was to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters.

* Corresponding author. Mailing address: Department of Chemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030. Phone: (619) 594-5575. Fax: (619) 594-4634. E-mail: wstumph{at}sciences.sdsu.edu.




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