Nucleic Acids Research, 2000, Vol. 28, No. 14 2771-2778
© 2000 Oxford University Press
Similarities and differences in the conformation of protein–DNA complexes at the U1 and U6 snRNA gene promoters
Department of Chemistry and Molecular Biology Institute, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA
Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. In the fruit fly Drosophila melanogaster the RNA polymerase specificity of the snRNA genes is determined by a few nucleotide differences within the proximal sequence element (PSE), a conserved sequence located ~40–65 bp upstream of the transcription start site. The PSE is essential for transcription of both RNA polymerase II-transcribed and RNA polymerase III-transcribed snRNA genes and is recognized in Drosophila by a multi-subunit protein factor termed DmPBP. Previous studies that employed site-specific protein–DNA photocrosslinking indicated that the conformation of the DNA–protein complex is different depending upon whether DmPBP is bound to a U1 or U6 PSE sequence. These conformational differences of the complex probably represent an early step in determining the selection of the correct RNA polymerase. We have now obtained evidence that DmPBP modestly bends the DNA upon interacting with the PSE and that the direction of DNA bending is similar for both the U1 and U6 PSEs. Under the assumption that DmPBP does not significantly twist the DNA, the direction of the bend in both cases is toward the face of the DNA helix contacted by the 45 kDa subunit of DmPBP. Together with data from partial proteolysis assays, these results indicate that the conformational differences in the complexes of DmPBP with the U1 and U6 PSEs more likely occur at the protein level rather than at the DNA level.
* To whom correspondence should be addressed. Tel: +1 619 594 5575; Fax: +1 619 594 4634; Email: wstumph@sciences.sdsu.edu
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