Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

抗人Elp3 的N末端抗体制备及在染色质免疫沉淀中的应用

人 Elp3（human elongator protein 3, hElp3）具有组蛋白乙酰转移酶活性,是与延伸中的 RNA 聚合酶Ⅱ结合的 elongator 复合物的催化亚基,可参与组蛋白的乙酰化修饰与基因的转录延伸. Elp3 及其复合物功能异常与人类多种疾病相关. 为运用染色质免疫沉淀等手段深入研究 Elp3 功能,PCR 法克隆 pYES2-hElp3 质粒中编码 hElp3的N端亲水区段（1～69 氨基酸残基）,构建原核表达载体 pMXB10-hElp3-210,经 IPTG 诱导和几丁质柱纯化后,免疫兔制备多克隆抗体. ELISA 检测显示,该抗体有较高的效价(不低于1∶2　500).免疫印迹实验结果表明,该抗体可与纯化的及 HeLa 细胞中的 hElp3 蛋白特异性结合.运用该抗体对转入 elp3Δ菌株的人 Elp3 的染色质免疫沉淀实验结果表明,人 Elp3 可参与酵母 SSA3 基因的转录调控,这可能是人Elp3 能够部分补偿酵母 SSA3 基因延迟表达缺陷的原因.