Schroeder H W, Liarakos C D, Gupta R C, Randerath K, O'Malley B W
Biochemistry. 1979 Dec 25;18(26):5798-808. doi: 10.1021/bi00593a009.
The region of the ovalbumin messenger ribonucleic acid (mRNAov) molecule bound to the 40S ribosomal subunit and its associated initiation factors in the wheat germ cell-free translation system were isolated and characterized. Two mRNAov fragments, 87 and 92 nucleotides in length, were protected from T1 ribonuclease digestion by binding of guanosine 5',beta,gamma-methylenetriphosphate and were shown by hybridization and fingerprint mapping to be derived from the 5' end of mRNAov. Both these mRNAov fragments were of sufficient length to contain both the cap structure and the AUG initiation codon. Four T1-resistant oligonucleotides, prepared by direct digestion of mRNAov with T1 ribonuclease were also found to bind to the wheat germ 40S ribosomal subunit. Nucleotide sequence analysis of these oligonucleotides revealed (1) that they were not a subset of the ribosome binding fragments described above, (2) that they were derived from within the mRNAov molecule (one from within the coding region and three from the noncoding region at the 3' end of the mRNAov molecule), and (3) that three of the four mRNAov nucleotides contained 3'-terminal AUG trinucleotides. These data suggested that features of the mRNAov molecule in addition to the nucleotide sequence might be important in specifying the correct ribosome binding site for the initiation of protein synthesis. The amount of mRNAov bound to the wheat germ 40S ribosomal subunit in a preinitiation complex was found to vary inversely with the potassium ion concentration. Lowering the potassium concentration to levels suboptimal for translation also resulted in the protection of larger fragments of the mRNAov molecule derived from the same 5'-end region as the ribosome binding fragments described above. The ability of the cap analogue 7-methylguanosine 5'-phosphate (m7G5'p) to reduce the amount of mRNAov bound to the wheat germ 40S ribosomal subunit was found to depend directly on thepotassium concentration. Interestingly, the effects of potassium on the amount of mRNAov bound in a preinitiation complex and the inhibition of this binding by m7G5'p could be observed by changing the potassium concentration after binding had occurred. These data suggested that the interaction between the wheat germ 40S ribosomal subunit and mRNAov was very sensitive to the ionic environment.
在麦胚无细胞翻译系统中，分离并鉴定了与40S核糖体亚基及其相关起始因子结合的卵清蛋白信使核糖核酸（mRNAov）分子区域。两个长度分别为87和92个核苷酸的mRNAov片段，通过与鸟苷5'，β，γ-亚甲基三磷酸结合而免受T1核糖核酸酶的消化，并通过杂交和指纹图谱显示它们源自mRNAov的5'端。这两个mRNAov片段的长度足以同时包含帽结构和AUG起始密码子。通过用T1核糖核酸酶直接消化mRNAov制备的四个抗T1寡核苷酸也被发现可与麦胚40S核糖体亚基结合。对这些寡核苷酸的核苷酸序列分析表明：（1）它们不是上述核糖体结合片段的一个子集；（2）它们源自mRNAov分子内部（一个源自编码区，三个源自mRNAov分子3'端的非编码区）；（3）四个mRNAov核苷酸中的三个含有3'-末端AUG三核苷酸。这些数据表明，除核苷酸序列外，mRNAov分子的特征对于确定蛋白质合成起始的正确核糖体结合位点可能也很重要。发现在起始前复合物中与麦胚40S核糖体亚基结合的mRNAov量与钾离子浓度呈反比。将钾浓度降低到不利于翻译的水平，也会导致源自与上述核糖体结合片段相同5'-末端区域的mRNAov分子的更大片段受到保护。发现帽类似物7-甲基鸟苷5'-磷酸（m7G5'p）减少与麦胚40S核糖体亚基结合的mRNAov量的能力直接取决于钾浓度。有趣的是，通过在结合发生后改变钾浓度，可以观察到钾对起始前复合物中结合的mRNAov量的影响以及m7G5'p对这种结合的抑制作用。这些数据表明，麦胚40S核糖体亚基与mRNAov之间的相互作用对离子环境非常敏感。
