Dontsova O, Tishkov V, Dokudovskaya S, Bogdanov A, Döring T, Rinke-Appel J, Thamm S, Greuer B, Brimacombe R
Department of Chemistry, Moscow State University, Russia.
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4125-9. doi: 10.1073/pnas.91.10.4125.
A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with T7 polymerase using a mixture of [alpha-32P]UTP and 4-thio-UTP, yielding a transcript in which approximately 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.
通过PCR从携带5S互补序列的M13克隆中制备了一个包含与T7启动子相连的大肠杆菌5S rDNA序列的DNA片段。使用[α-32P]UTP和4-硫代UTP的混合物，用T7聚合酶转录该DNA，产生一种转录本，其中约18%的尿苷残基被硫代尿苷随机取代。这种修饰的5S RNA可以有效地重组成50S核糖体亚基或70S功能复合物。对重组颗粒在波长高于300 nm的条件下进行辐照，并鉴定交联的核糖体组分。在修饰的5S RNA和23S RNA之间可重复观察到高产率的交联，涉及5S RNA的U-89残基（在螺旋IV的环端）与23S RNA的2477核苷酸在螺旋89的环端交联，紧邻肽基转移酶“环”。基于这一结果，并结合早期的免疫电子显微镜数据，我们提出了一个5S RNA在50S亚基中取向的模型。
