Göringer H U, Bertram S, Wagner R
J Biol Chem. 1984 Jan 10;259(1):491-6.
The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.
利用化学试剂乙二醛（α-酮-β-乙氧基丁醛）修饰可及鸟苷，研究了来自大肠杆菌的70 S核糖体中5 S RNA的结构。将游离70 S核糖体的5 S RNA修饰模式与多聚尿苷酸（poly(U)）编程核糖体的修饰模式进行了比较，在多聚尿苷酸编程核糖体中，tRNA已结合到A位点和P位点。在脱酰基tRNA存在的情况下，利用延伸因子Tu和GTP通过酶促作用实现与核糖体A位点的结合，脱酰基tRNA会阻断核糖体P位点。在部分核糖核酸酶T1水解并在测序凝胶上分离水解产物后，鉴定出修饰的鸟苷。tRNA与核糖体的结合导致5 S RNA鸟苷G-41以及在一定程度上G-44受到强烈保护，不被乙二醛修饰。有限的核糖核酸酶T1水解模式为存在一种不同于已知5 S RNA A型和B型的5 S RNA构象提供了证据，已知的5 S RNA A型和B型以其凝胶电泳迁移率为特征。讨论了5 S RNA对tRNA与核糖体结合的重要性。
