Chen J K, Franke L A, Hixson S S, Zimmermann R A
Biochemistry. 1985 Aug 27;24(18):4777-84. doi: 10.1021/bi00339a011.
The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.
用三种不同长度的光亲和试剂对来自大肠杆菌的tRNA1Arg第32位的2-硫代胞苷残基进行特异性修饰，通过照射使相应的N-乙酰精氨酰-tRNA1Arg衍生物与大肠杆菌70S核糖体的P位点交联。共价连接取决于多核苷酸模板的存在以及暴露于适当波长的光。由于光解作用，4%至6%的非共价结合tRNA与核糖体交联，共价复合物中精氨酸对嘌呤霉素的反应性证实了其与P位点的结合。在低Mg2+浓度下通过蔗糖梯度沉降分析照射后的核糖体，结果表明在所有情况下tRNA仅与30S亚基结合。其中两种N-乙酰精氨酰-tRNA1Arg衍生物主要与核糖体蛋白结合，而第三种主要与16S RNA交联。对后一种复合物进行部分RNase消化表明，tRNA已与rRNA分子的3'末端三分之一结合。此外，tRNA-rRNA键显示易被羟胺和巯基乙醇裂解。
