Boni I V, Isaeva D M, Musychenko M L, Tzareva N V
M.M. Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow.
Nucleic Acids Res. 1991 Jan 11;19(1):155-62. doi: 10.1093/nar/19.1.155.
Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome.
已知核糖体蛋白S1在翻译起始过程中发挥重要作用，它直接参与30S核糖体颗粒对mRNA的识别和结合。通过一种基于紫外线照射诱导S1与mRNA高效交联的特殊开发方法，我们在起始前复合物中的几种噬菌体RNA上鉴定出了S1结合位点。Qβ和fr RNA上S1的作用靶点位于外壳蛋白基因上游，且含有寡聚（U）序列。就Qβ RNA而言，该S1结合位点与Qβ复制酶的S位点以及二元复合物中的S1结合位点重叠。类似的富含U的序列代表细菌mRNA上的S1结合位点是合理的。为了验证这一想法，我们使用了通过T7启动子/RNA聚合酶系统体外制备的大肠杆菌单链结合蛋白（ssb）mRNA。通过足迹法、酶足迹法和紫外线交联法，我们表明ssb mRNA与30S核糖体的结合依赖于S1。发现SD结构域之前的寡聚（U）序列是S1的作用靶点。我们提出，由SD区域上游富含嘧啶的序列代表的S1结合位点，作为核糖体识别mRNA所涉及的决定因素。
