Jay G, Kaempfer R
J Biol Chem. 1975 Aug 10;250(15):5742-8.
Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.
在30 S亚基与甲硫氨酸- tRNA以及除R17噬菌体RNA外的所有其他用于蛋白质合成起始的成分一起温育过程中，会形成30 S核糖体亚基与甲硫氨酸- tRNA的复合物。这些复合物作为信使RNA结合的中间体，这一发现直接证明了：在添加R17 RNA后，预先形成的甲硫氨酸- RNA - 30 S复合物中的甲硫氨酸- tRNA会优先进入甲硫氨酸- tRNA - 30 S - R17 RNA复合物。另一方面，将30 S核糖体亚基与R17 RNA以及除甲硫氨酸- tRNA外的所有其他用于起始的成分一起温育，不会产生随后可作为甲硫氨酸- tRNA结合功能中间体的30 S - R17 RNA复合物：添加甲硫氨酸- tRNA时不会形成甲硫氨酸- tRNA - 30 S - R17 RNA复合物，并且特异性抑制剂会阻止R17 RNA与30 S亚基的进一步结合。这些实验得出了起始序列中明确无误的事件顺序，即甲硫氨酸- tRNA与小核糖体亚基的结合必须在信使RNA能够结合并正确定相之前发生。甲硫氨酸- tRNA与60 S亚基之间的复合物与游离成分处于快速平衡状态，在37摄氏度时半衰期小于2分钟。这就解释了为什么在蔗糖梯度中检测不到这种复合物，除非它们首先用戊二醛固定。然而，R17 RNA的附着会导致形成一种大大稳定的甲硫氨酸- tRNA - 30 S - R17 RNA复合物；该复合物中的甲硫氨酸- tRNA与游离甲硫氨酸- tRNA的交换非常缓慢。起始因子IF - 3在起始过程中有两个功能。第一个功能是指导信使RNA与30 S -甲硫氨酸- tRNA复合物的结合。当在ApUpG三联体上形成起始复合物时，不需要这个功能，在这种情况下会检测到IF - 3的第二个功能，即为起始提供游离的30 S亚基。IF - 3直接与R17 RNA结合的能力可能与其在信使RNA识别中的需求有关。然而，由于IF - 3对30 S亚基的亲和力比对R17 RNA的亲和力更大，似乎IF - 3的识别功能是在IF - 3与30 S亚基结合时表达的。
