Conformation states of Xenopus transcription factor IIIA.
The conformation of Xenopus transcription factor IIIA (TFIIIA) free in solution, bound to 5S RNA in the 7S particle, depleted of zinc, or bound to plasmid DNA was analyzed by (1) trypsin digestion and electrophoretic analysis of proteolytic fragments or (2) measurement of the fluorescence of TFIIIA mildly derivatized with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS). TFIIIA free or complexed with 5S RNA has a similar conformation as judged (a) by trypsin-dependent generation of similar metastable 20-kDa domains (corresponding to the N-terminal half of the protein) or (b) by the negligible change in AEDANS-TFIIIA fluorescence when free or bound to 5S RNA. When TFIIIA binds plasmid DNA, its N-terminal half becomes hypersensitive to trypsin digestion, indicating a structural change in this region of the protein upon interaction with DNA. Quenching of AEDANS-TFIIIA fluorescence is observed upon interaction of the protein with plasmid DNA, a result also indicative of a conformational change upon protein-DNA interaction. Removal of zinc from TFIIIA by EDTA chelation results in (a) increased proteolysis of this 20-kDa domain, indicating a structural change in the N-terminal half of the protein upon zinc removal, and (b) large enhancement of AEDANS-TFIIIA fluorescence. EDTA chelation of TFIIIA bound to 5S RNA in the 7S particle, a procedure which does not deplete all zinc from the protein, neither increases the trypsin sensitivity of the 20-kDa domain nor alters appreciably the fluorescence of AEDANS-TFIIIA. These results indicate that zinc is involved in maintaining the native conformation of at least the N-terminal half of the protein.
Hanas
J S
JS
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Duke
A L
AL
Gaskins
C J
CJ
D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D008970:Molecular Weight; D009282:Naphthalenesulfonates / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012996:Solutions; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D012996:Solutions; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; C006421:1,5-I-AEDANS; D004492:Edetic Acid; D014357:Trypsin