Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA.
A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.
Lee
D K
DK
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Evans
R K
RK
Blanco
J
J
Gottesfeld
J
J
Johnson
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001483:Base Sequence; D015342:DNA Probes; D004275:DNA, Ribosomal / Q000378:metabolism* / Q000528:radiation effects; D003856:Deoxyuracil Nucleotides; D005091:Exons; D007438:Introns; D008969:Molecular Sequence Data; D010777:Photochemistry; D011487:Protein Conformation; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D013698:Templates, Genetic; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014981:Xenopus; D016335:Zinc Fingers
D001386:Azides; D015342:DNA Probes; D004275:DNA, Ribosomal; D003856:Deoxyuracil Nucleotides; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; C048865:5-azido-2'-deoxyuridine 5'-triphosphate