Han M K, Cyran F P, Fisher M T, Kim S H, Ginsburg A
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Aug 15;265(23):13792-9.
The release of Zn2+ from transcription factor IIIA (TFIIIA) was examined with the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) in the absence and presence of p-hydroxymercuriphenylsulfonate (PMPS). With 0.5 mM PAR, approximately 5 eq of Zn2+ were released from TFIIIA, but no Zn2+ release was detected from the 7 S ribonucleoprotein. The PMPS-promoted Zn2+ release from TFIIIA was 8.7 +/- 0.4 eq of Zn2+ of which approximately 4 eq of Zn2+ rebound to TFIIIA upon displacement of the mercurial with excess 2-mercaptoethanol. These results suggest that at least two affinity classes of Zn2+ binding sites exist in TFIIIA, one of which is released to 0.5 mM PAR in the absence of PMPS. Also, 18 of the 23 cysteine residues of TFIIIA reacted with 5,5'-dithiobis-(2-nitrobenzoic acid). The kinetic data of PAR and 5,5'-dithiobis-(2-nitrobenzoic acid) reactions with TFIIIA were similar, and the spectral changes were characterized by at least three exponential terms. Both TFIIIA and the 7 S particle were reacted with the thiol-specific fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS). Complete trypsin hydrolysis followed by reverse-phase high pressure liquid chromatography analysis of peptide mixtures showed only one fluorescent peak from the AEDANS-labeled 7 S particle whereas numerous fluorescent peaks were observed with AEDANS-labeled TFIIIA. This further indicates exposure of cysteine residues from Zn2+ binding domains in TFIIIA. Cys287 was identified as the site of modification by amino acid sequencing of the isolated fluorescent peptide from the derivatized 7 S particle. Limited papain cleavage of the AEDANS-labeled 7 S particle indicated that the modified cysteine is located within a 34-kDa TFIIIA fragment. Gel retardation and transcription assays showed that TFIIIA, which had been purified from the AEDANS-labeled 7 S particle, was capable of binding to the internal control region of 5 S RNA gene and retained transcription activity. Thus, Zn2+ binding domains and all but 1 cysteine residue are buried in the 7 S particle, thereby facilitating site-specific labeling of TFIIIA.
