Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters.
The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.
Kim
Mun Kyoung
MK
Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-1030, USA.
Kang
Yoon Soon
YS
Lai
Hsien-Tsung
HT
Barakat
Nermeen H
NH
Magante
Deodato
D
Stumph
William E
WE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009711:Nucleotides; C483821:Pbp45 protein, Drosophila; C534011:Pbp49 protein, Drosophila; C534010:Pbp95 protein, Drosophila; D021122:Protein Subunits; D012342:RNA, Small Nuclear; C106703:SNAPC protein complex, human; D014157:Transcription Factors; C405483:U1 small nuclear RNA; C405486:U6 small nuclear RNA