Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site.
Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site.
复制标题
未修饰的乙酰缬氨酰-tRNA 在核糖体 P 位点处与 16S RNA 发生光化学交联。
作者：
I. Schwartz;J. Ofengand
乙酰戊酰-、乙酰苯丙酰-和甲酰基甲基- trna在其4-硫脲残基上被衍生化，具有光亲和标记对叠氮苯酰溴，它们被非酶结合到盐洗核糖体上。根据与嘌呤霉素的反应性判断，超过90%的结合在P位点。随后trna -核糖体复合物的辐照（大于310 nm）导致仅乙酰酰- trna与30S亚基的共价连接。仅与16S RNA结合，交联效率为13—15%。预先用嘌呤霉素处理可抑制90%的共价连接，表明共价连接反应发生在P位点。交联需要辐照和mRNA，但不依赖于tRNA中光亲和探针的存在。用对叠氮苯酰溴或未修饰的乙酰戊基trna修饰4-硫脲的trna表现出与光亲和探针修饰的乙酰戊基trna相同的交联行为。此外，即使是经过H2O2处理去除4-硫脲的乙酰戊基tRNA，其活性也与未修饰的tRNA在数量上相同。这些结果首次证明了tRNA与核糖体的直接光化学交联。
Acetylvalyl-, acetylphenylalanyl-, and formylmethionyl-tRNA which were derivatized at their 4-thiouridine residues with the photoaffinity label, p-azidophenacyl bromide, were nonenzymatically bound to salt-washed ribosomes. More than 90% of the binding was to the P site as judged by reactivity with puromycin. Subsequent irradiation (greater than 310 nm) of the tRNA-ribosome complexes resulted in the covalent linking of only the acetylvalyl-tRNA to the 30S subunit. Attachment was solely to the 16S RNA with an efficiency of cross-linking of 13--15%. Covalent linking was 90% inhibited by prior treatment with puromycin, showing that the covalent linking reaction had taken place at the P site. Cross-linking required irradiation and mRNA but was not dependent on the presence of the photoaffinity probe in the tRNA. tRNAs whose 4-thiouridine had been modified with unreactive analogues of p-azidophenacyl bromide or unmodified acetylvalyl-tRNA exhibited the same cross-linking behavior as photoaffinity probe-modified acetylvalyl-tRNA. Furthermore, even acetylvalyl-tRNA whose 4-thiouridine had been removed by treatment with H2O2 was quantitatively as active as unmodified tRNA. These results provide the first demonstration of direct photochemical cross-linking of tRNA to ribosomes.