[大肠杆菌核糖体供体tRNA结合中心附近的光亲和修饰研究]

[Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center].

作者信息

Bausk E V, Graĭfer D M, Karpova G G

出版信息

Mol Biol (Mosk). 1985 Mar-Apr;19(2):545-52.

Abstract

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.

摘要

利用在鸟嘌呤残基的N7原子上带有芳基叠氮基团的苯丙氨酸tRNA的光反应性衍生物（叠氮基-tRNA），研究了大肠杆菌核糖体在供体tRNA结合（P）位点附近的亲和标记。对70S核糖体·聚（U）·叠氮基-tRNA（P位点）和70S核糖体·聚（U）·叠氮基-tRNA（P位点）·苯丙氨酸-tRNA（A位点）复合物进行紫外线照射，导致叠氮基-tRNA与核糖体共价结合，两个亚基均被标记。在这两种情况下，30S亚基的修饰程度是50S亚基的两倍。结果表明，当A位点空闲时，位于P位点的叠氮基-tRNA标记了蛋白质S9、S11、S12、S13、S21以及L14、L27、L31。当A位点被苯丙氨酸-tRNA占据时，位于P位点的叠氮基-tRNA标记了蛋白质S11、S12、S13、S14、S19、L32/L33以及可能的L23、L25。通过比较A位点空闲或被占据时标记的蛋白质组，得出结论：位于核糖体A位点的氨酰-tRNA会影响P位点去酰化tRNA的排列。所获得的数据表明，蛋白质S5、S19、S20以及L24、L33与对tRNA三级结构形成重要的鸟嘌呤残基相互作用。