Browning K S, Lax S R, Humphreys J, Ravel J M, Jobling S A, Gehrke L
Department of Chemistry, University of Texas, Austin 78712-1096.
J Biol Chem. 1988 Jul 15;263(20):9630-4.
The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.
在小麦胚芽的S30提取物以及小麦胚芽分级分离系统中，测定了苜蓿花叶病毒（AMV）RNA 4、大麦α-淀粉酶（BαA）mRNA以及两种嵌合mRNA（AMV 4-BαA和BαA-AMV 4，其中两种mRNA的5'前导序列互换）的翻译效率，在该分级分离系统中，翻译可依赖于起始因子（eIF）3、4A、4F或4G。在S30系统中，AMV RNA 4和嵌合mRNA AMV 4-BαA的翻译效率比BαA mRNA和嵌合mRNA BαA-AMV 4高得多。当向S30系统中添加大量纯化的eIF-3、eIF-4A、eIF-4F和eIF-4G时，BαA和BαA-AMV 4 mRNA的翻译效率与AMV RNA 4和AMV 4-BαA mRNA相同。这些发现表明，含有BαA前导序列的mRNA高效翻译需要一种或多种起始因子（eIF-3、eIF-4A、eIF-4F和eIF-4G）的量更高。在分级分离系统中对翻译所需起始因子量的测定表明，与BαA mRNA相比，AMV RNA 4所需的eIF-3、eIF-4A、eIF-4F和eIF-4G量低2至4倍。用AMV RNA 4的前导序列替换BαA的前导序列可降低所需的eIF-4A、eIF-4G和eIF-3量，但不影响所需的eIF-4F量。用BαA mRNA的前导序列替换AMV RNA 4的前导序列可增加所需的eIF-4F、eIF-4G和eIF-3量，但不影响所需的eIF-4A量。这些数据有力地表明，所需因子的量不仅受5'前导序列本身的影响，还受5'前导序列与起始密码子3'端mRNA区域之间相互作用的影响。
