在缺乏转运RNA的情况下检测大肠杆菌核糖体在翻译起始位点的结合情况。

Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA.

作者信息

Hartz D, McPheeters D S, Green L, Gold L

机构信息

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.

出版信息

J Mol Biol. 1991 Mar 5;218(1):99-105. doi: 10.1016/0022-2836(91)90876-8.

DOI:10.1016/0022-2836(91)90876-8

PMID:2002510

Abstract

Binary complexes between messenger RNA and E. coli ribosomes were examined. A ribosome-mRNA binary complex on T4 gene 32 mRNA withstood inhibition by antibodies against ribosomal protein S1. Anti-S1 blocks ternary complex formation, as measured by "extension inhibition" or "toeprinting" analysis, only when preincubated with ribosomes prior to mRNA addition and not when anti-S1 was added after preincubation of ribosomes and mRNA. The ribosome was directly localized in a binary complex on two translation initiation sites by toeprinting analysis. In the absence of tRNA the ribosome halted cDNA synthesis by reverse transcriptase close to the Shine and Dalgarno sequence. Binary complex formation was inhibited by an oligodeoxynucleotide competitor of the Shine and Dalgarno sequence.

摘要

对信使核糖核酸（mRNA）与大肠杆菌核糖体之间的二元复合物进行了研究。T4基因32 mRNA上的核糖体-mRNA二元复合物能抵抗针对核糖体蛋白S1的抗体的抑制作用。通过“延伸抑制”或“足迹法”分析测定，抗S1只有在mRNA添加之前与核糖体预孵育时才会阻断三元复合物的形成，而在核糖体和mRNA预孵育后添加抗S1时则不会。通过足迹法分析，核糖体直接定位于两个翻译起始位点的二元复合物中。在没有转运核糖核酸（tRNA）的情况下，核糖体通过逆转录酶使互补脱氧核糖核酸（cDNA）合成在靠近夏因-达尔加诺序列处停止。夏因-达尔加诺序列的寡脱氧核苷酸竞争者会抑制二元复合物的形成。