IDENTIFICATION OF FEATURES IN 5' TERMINAL FRAGMENTS FROM REOVIRUS MESSENGER-RNA WHICH ARE IMPORTANT FOR RIBOSOME BINDING
IDENTIFICATION OF FEATURES IN 5' TERMINAL FRAGMENTS FROM REOVIRUS MESSENGER-RNA WHICH ARE IMPORTANT FOR RIBOSOME BINDING
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DOI：
10.1016/0092-8674(78)90150-2
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发表时间：
1978-01-01
期刊：
影响因子：
64.5
通讯作者：
SHATKIN, AJ
中科院分区：
文献类型：
--
作者：
KOZAK, M;SHATKIN, AJ
用呼肠孤病毒m RNA或已知序列的5‘’末端片段进行了四种类型的实验，以确定m RNA中似乎对与核糖体形成起始复合体很重要的特征。与80S起始复合体相比，40S起始复合体保护的RNA片段(包括5‘端m7G)明显大于80S起始复合体。每个80S保护序列都有一个AUG密码子，并且是来自相同消息的40S保护序列的子集。当测试40S和80S保护的片段与核糖体重新结合的能力时，80S保护的片段显示出相当低的结合能力，这意味着由40S起始复合体保护的额外序列有助于核糖体的附着。然而，小麦胚核糖体在每个呼肠孤病毒mRNA5‘端选择相同的起始点，无论消息上是否有m7G。这是通过比较甲基化和未甲基化的RNA获得的核糖体保护区的指纹来证明的。因此，m7G对引发络合物形成的贡献是定量的，而不是定性的。从几个呼肠孤病毒信息中分离的5‘’末端片段经T1RNase限制性消化后，产生了一系列较小的片段，并分析了这些片段与核糖体重新结合的能力。保留5‘端密码子而不保留AUG密码子的长达30个核苷酸的部分消化产物不能与核糖体稳定结合，但分析的每一个含有AUG的片段都能形成起始复合体。然而，通过去除包含AUG的3‘侧的5’‘末端区域(包括帽)或包括编码区开始的序列，降低了某些包含AUG的片段的结合效率。三核苷酸Aug能抑制mRNA与核糖体之间的复合体形成，但不能被其他各种寡核苷酸所抑制。虽然这种抑制是特异性的，但适度抑制80S复合体的形成需要大量的三核苷酸，同样浓度的AUG对40S起始复合体的形成没有抑制作用。
Four types of experiments were carried out with reovirus mRNA or with 5'' terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNA, 40S initiation complexes were previously shown to protect a significantly larger segment of the RNA (including the 5'' terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the extra sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5'' terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated vs. unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNase digestion of isolated 5'' terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5'' cap but not the AUG codon were unable to associate stably with ribosomes, but every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of the 5'' terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3'' side of the AUG. Complex formation between mRNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.