Napthine Sawsan, Vidakovic Marijana, Girnary Roseanne, Namy Olivier, Brierley Ian
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
EMBO J. 2003 Aug 1;22(15):3941-50. doi: 10.1093/emboj/cdg365.
Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.
核糖体移码信号存在于原核生物和真核生物的移动遗传元件、病毒及细胞基因中。通常它们由一个滑序列(X XXY YYZ,移码发生于此)和一个刺激性mRNA元件组成。在此我们研究了宿主翻译环境以及滑序列解码tRNA的身份对移码机制的影响。通过在大肠杆菌中以及在内源tRNA缺失并补充了原核或真核tRNA群体的麦胚提取物中表达候选信号,我们发现,当在核糖体A位点解码AAG时,大肠杆菌tRNA(Lys)在原核和真核核糖体中均促进了一种非常不寻常的单tRNA滑移事件。该事件似乎并不需要相邻P位点tRNA的滑移,尽管其身份具有影响力。相反,天冬酰胺tRNA在任一系统中均促进了双滑移事件。因此,tRNA本身是选择单或双tRNA滑移机制的主要决定因素。我们还首次表明,原核tRNA(Asn)并非天生“不易滑移”,在真核翻译系统背景下会诱导高效的移码。