Shang Z, Liao Y D, Wu F Y, Wu C W
Department of Pharmacological Sciences, School of Medicine, State University of New York, Stony Brook 11794.
Biochemistry. 1989 Dec 12;28(25):9790-5. doi: 10.1021/bi00451a037.
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.
非洲爪蟾转录因子IIIA（TFIIIA）含有两个紧密结合的固有锌离子，通过用对（羟基汞）苯磺酸盐（PMPS）或焦碳酸二乙酯（DEP）处理可释放这些锌离子，这通过金属显色指示剂4-（2-吡啶偶氮）间苯二酚（PAR）进行监测。通过体外5S RNA基因转录试验检测到的DEP对TFIIIA的失活与组氨酸残基的修饰程度和锌离子释放相关。与PMPS反应后，7S颗粒解离成游离的TFIIIA和5S RNA。这种解离可能与半胱氨酸残基的修饰程度以及锌离子释放相关。通过添加过量的硫醇试剂可使7S颗粒的解离逆转。然而，EDTA可抑制这种可逆性，表明锌离子是TFIIIA与5S RNA结合所必需的。在存在PMPS或DEP修饰的TFIIIA或锌离子耗尽的TFIIIA的情况下，疏水探针8-苯胺基萘磺酸盐的最大荧光发射蓝移了30 nm，而在存在7S颗粒或TFIIIA的情况下仅观察到小于10 nm的蓝移。这些结果表明，TFIIIA中的两个锌离子与半胱氨酸和组氨酸残基配位，并且是维持TFIIIA正确构象所必需的。
