Glanville N, Ranki M, Morser J, Kääriäinen L, Smith A E
Proc Natl Acad Sci U S A. 1976 Sep;73(9):3059-63. doi: 10.1073/pnas.73.9.3059.
The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.
在体外，用起始tRNA上的甲酰-[35S]甲硫氨酸标记了响应于塞姆利基森林病毒42S和26S RNA而合成的蛋白质。一种与病毒衣壳蛋白迁移率相同的蛋白质在26S RNA的指导下被标记，用胰蛋白酶消化后仅检测到一种标记肽。用链霉蛋白酶进一步消化产生二肽fMet-AsN。在42S RNA指导的产物中发现了几种标记的多肽，其分子量高达150,000。然而，该产物的胰蛋白酶消化仅产生一种甲酰甲硫氨酰标记的肽，其迁移率与26S RNA指导的肽不同。用链霉蛋白酶进一步消化得到单一二肽fMet-Ala。这表明高达150,000道尔顿的非结构蛋白可能从42S RNA上的一个起始位点合成。在所用条件下未检测到从42S RNA上的内部起始位点开始的翻译，该位点与26S RNA上的起始位点相当。在其他病毒RNA（例如雀麦花叶病毒、烟草花叶病毒和多瘤病毒19S RNA）中也检测到了类似无活性的内部起始位点，这表明，尽管真核mRNA可以包含多个蛋白质合成起始位点，但在体外只有更靠近5'末端的位点是活跃的。
