Bovia F, Bui N, Strub K
Département de Biologie Cellulaire, Université de Genève, Switzerland.
Nucleic Acids Res. 1994 Jun 11;22(11):2028-35. doi: 10.1093/nar/22.11.2028.
The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.
新生多肽链向内质网的靶向作用是由一种细胞质核糖核蛋白——信号识别颗粒（SRP）介导的。SRP的9kD（SRP9）和14kD（SRP14）亚基是赋予该颗粒延伸阻滞活性所必需的。SRP9和SRP14形成一个异二聚体，它特异性地结合SRP RNA。我们构建了cDNA，其编码由17个氨基酸肽连接的两种可能排列方式的包含SRP9和SRP14序列的单条多肽链。我们发现这两种融合蛋白都作为折叠成异二聚体样结构的单体分子特异性地结合SRP RNA。我们的结果证实了先前的假设，即真正的异二聚体以等摩尔比结合SRP RNA。此外，这两种融合蛋白都赋予缺乏该功能的SRP（-9/14）延伸阻滞活性，并且一种融合蛋白在转位测定中可以在功能上替代异二聚体。因此，两种蛋白质正常的N端和C端在折叠、RNA结合以及介导生物学活性方面没有重要作用。将异二聚体复合物表达为单条多肽链的可能性便于在体内和体外分析其功能和结构。
