Zinc release from Xenopus transcription factor IIIA induced by chemical modifications

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.