Sleat D E, Hull R, Turner P C, Wilson T M
Department of Virus Research, John Innes Institute and Agricultural and Food Research Council Institute of Plant Science Research, Norwich, England.
Eur J Biochem. 1988 Jul 15;175(1):75-86. doi: 10.1111/j.1432-1033.1988.tb14168.x.
Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.
烟草花叶病毒RNA（普通株系）5'-前导序列（ω）的顺式作用衍生物（ω'）可增强外源mRNA的翻译。为了解释这种效应，我们进行了多项体外实验。1. 包括ω'在内的各种5'-末端序列的存在，并未显著增加小麦胚芽提取物中氯霉素乙酰转移酶（CAT）或新霉素磷酸转移酶（NPTII）mRNA的半衰期。此外，与ω'无关的长前导序列在体外也未增强NPTII mRNA的表达。2. 使用在稀疏霉素存在下孵育的CAT或NPTII mRNA，检测了包括ω'在内的几种前导序列与80S（小麦胚芽）核糖体形成多个起始复合物的能力。二体复合物的形成与5'-前导序列增强翻译的能力无关。3. 当存在5'-近端ω'序列时，小麦胚芽提取物和信使依赖性兔网织红细胞裂解物中CAT mRNA的表达受盐浓度增加的抑制作用较小。当CAT mRNA加帽时，这种效应不太明显。相反，在高盐浓度下，加帽对具有5'-近端ω'序列的mRNA的刺激作用较小。这些数据表明，ω'和帽至少部分地通过类似机制增强翻译。我们提出，这两个特征都减少了RNA二级结构，从而使5'末端更容易被40S核糖体亚基扫描和/或与相关起始因子相互作用。基于计算机的我们的SP6 RNA聚合酶转录本序列二级结构分析支持了这一结论。来自雀麦花叶病毒RNA 3、苜蓿花叶病毒RNA 4以及芜菁黄花叶病毒、劳氏肉瘤病毒或烟草花叶病毒（番茄株系）基因组RNA的5'前导序列在真核系统中增强mRNA翻译的能力，也可能与其各自的二级结构相关。一种不同的机制可能解释了在大肠杆菌或大肠杆菌无细胞系统中mRNA表达的ω'依赖性增强。
