Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.
Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.
复制标题
5S rRNA 的茎环 IV 靠近肽基转移酶中心。
DOI：
--
复制
发表时间：
1994
影响因子：
11.1
通讯作者：
Richard Brimacombett
中科院分区：
文献类型：
--
作者：
Olga Anatoljevna Dontsova;Vladimir Tishkov;Svetlana Dokudovskaya;Alexei Bogdanov;Thomas Doringt;Jutta Rinke;Sabine Thammt;Barbara Greuert;Richard Brimacombett
从携带5S-互补序列的M13克隆中，通过聚合酶链式反应(PCR)获得与T7启动子相连的大肠杆菌5S rDNA序列的DNA片段。用[α-32P]UTP和4-硫代UTP的混合物用T7聚合酶转录DNA，产生一个转录本，其中大约18%的尿苷残基被硫代尿苷随机取代。这种修饰后的5S RNA可以有效地重组为50S核糖体亚基或70S功能复合体。重组粒子在300 nm以上的波长照射，并鉴定了交联型核糖体成分。在修饰的5S RNA和23S RNA之间重复性地观察到一个高产量的交联物，包括5S RNA的残基U-89(在螺旋IV的环端)与23S RNA的2477核苷酸连接在螺旋89的环端，紧邻肽转移酶“环”。在这一结果的基础上，结合早期的免疫电子显微镜数据，我们提出了一个50S亚基中5S RNA的定位模型。
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.