MacKeen L A, Kahan L, Wahba A J, Schwartz I
J Biol Chem. 1980 Nov 10;255(21):10526-31.
Initiation factor-3 has been photochemically cross-linked to Escherichia coli 30 S ribosomal subunits by means of near-ultraviolet (> 285 nm) irradiation. The cross-linking was judged to be specific since noncovalent binding was required for subsequent cross-linking and was prevented by the presence of 0.5 M NH4Cl or 0.5 mM aurintricarboxylic acid. Covalent attachment reached its maximum level of 8.5 to 11% of the noncovalently bound IF-3 after 60 min of irradation. Cross-linking was unaffected by the presence of the photosensitizer, acetone (5 or 10%), but was reduced to 30 to 40% of its maximum level by addition of 5 mM dithiothreitol, a free radical scavenger. Analysis of the 14C-labeled IF-3 x 30 S subunit covalent complex revealed that the IF-3 was distributed between the protein and RNA of the subunits in a 3:1 ratio. The target proteins have been identified as S7, S11, S12, S18, and S21 by immunochemical techniques.
起始因子-3已通过近紫外光（>285纳米）照射与大肠杆菌30S核糖体亚基进行光化学交联。由于后续交联需要非共价结合，且0.5M氯化铵或0.5mM金精三羧酸的存在可阻止交联，因此判断该交联具有特异性。辐照60分钟后，共价结合达到非共价结合IF-3的8.5%至11%的最大水平。交联不受光敏剂丙酮（5%或10%）的影响，但通过添加5mM二硫苏糖醇（一种自由基清除剂）可将其降低至最大水平的30%至40%。对14C标记的IF-3×30S亚基共价复合物的分析表明，IF-3以3:1的比例分布在亚基的蛋白质和RNA之间。通过免疫化学技术已将靶蛋白鉴定为S7、S11、S12、S18和S21。
