Zhou L, Marzluf G A
Department of Biochemistry, Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus 43210, USA.
Biochemistry. 1999 Apr 6;38(14):4335-41. doi: 10.1021/bi982543f.
Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.
在粗糙脉孢菌中存在多种GATA因子，即识别共有GATA元件的锌指DNA结合蛋白。其中一种，即SRE，参与调控粗糙脉孢菌的铁代谢途径。在粗糙脉孢菌中，铁的转运由一些被称为铁载体的小环肽介导。铁载体合成途径受SRE负调控；功能缺失的sre突变菌株表现出铁载体的部分组成型合成。在本文所述的研究中，通过异核体试验和基因互补进一步证实了SRE的负功能。SRE被表达为一种GST融合蛋白。体外电泳迁移率变动分析（EMSA）表明，SRE能特异性结合含有GATA序列元件的DNA分子。sre基因表达的自我调节似乎是可能的，因为sre基因启动子本身含有GATA序列。在sre中引入突变，导致每个锌指中的氨基酸替换，这将破坏它们的功能。体外EMSA表明，SRE的N端和C端锌指均参与DNA结合。这一特征与脊椎动物的双锌指GATA因子不同。通过将突变的sre基因转化到sre rip突变体中进行的体内试验表明，在单锌指或双锌指发生突变的SRE在低铁条件下仍能保持其功能。相比之下，这些突变的SRE蛋白在高铁条件下无法发挥功能。我们的结果预测铁载体合成途径存在其他正调控或负调控因子。
