Skouv J, Schnier J, Rasmussen M D, Subramanian A R, Pedersen S
Institute of Microbiology, University of Copenhagen, Denmark.
J Biol Chem. 1990 Oct 5;265(28):17044-9.
To facilitate the study of the regulation of the rpsA gene, a translational fusion between the rpsA gene and the lacZ gene was constructed. Synthesis of the fusion protein was repressed about 10-fold when rpsA was supplied in trans on a multicopy plasmid. This repression is similar to the post-transcriptional regulation previously found for the wild type rpsA gene. Addition of purified protein S1 to a coupled in vitro transcription-translation system caused a specific reduction in the synthesis of the rpsA-lacZ fusion protein. Addition of various subdomain fragments of protein S1 to the coupled in vitro system showed that the N-terminal fragment, possessing the ribosome binding domain of protein S1, was able to repress the synthesis of the rpsA-lacZ fusion protein. In contrast, fragments from the C-terminal region, containing the nucleic acid binding domain of protein S1, were inactive in this repression. Induction of truncated rpsA genes, coding for either the N-terminal 101 or 329 amino acids caused a reduction in the synthesis of the chromosomally encoded protein S1, thus confirming in vivo that the N-terminal part of protein S1 represses rpsA expression.
为便于研究rpsA基因的调控,构建了rpsA基因与lacZ基因之间的翻译融合体。当rpsA通过多拷贝质粒反式提供时,融合蛋白的合成被抑制了约10倍。这种抑制类似于先前在野生型rpsA基因中发现的转录后调控。将纯化的蛋白S1添加到偶联的体外转录-翻译系统中,导致rpsA-lacZ融合蛋白的合成特异性减少。将蛋白S1的各种亚结构域片段添加到偶联的体外系统中表明,具有蛋白S1核糖体结合结构域的N端片段能够抑制rpsA-lacZ融合蛋白的合成。相反,来自C端区域的片段,包含蛋白S1的核酸结合结构域,在这种抑制中没有活性。编码N端101或329个氨基酸的截短rpsA基因的诱导导致染色体编码的蛋白S1的合成减少,从而在体内证实蛋白S1的N端部分抑制rpsA表达。