Epe B, Woolley P, Steinhäuser K G, Littlechild J
Eur J Biochem. 1982 Dec;129(1):211-9. doi: 10.1111/j.1432-1033.1982.tb07042.x.
Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyl and/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3' end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster-type energy transfer. The result for S4 was 6.0 nm (60 A) in the ribonucleoprotein complex and 5.6 nm (56 A) in the 30-S subunit, and for S17 6.3 nm (63 A) in the complex and 6.2 nm (62 A) in the subunit. There is no evidence for a major change in the relative disposition of the 3' and 5' ends of the 16-S RNA during formation of the 30-S subunit. Sources of error are discussed, including the question of multiple labelling. In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.
大肠杆菌核糖体蛋白S4和S17的巯基用乙酰氨基乙基-丹磺酰基和/或二甲基马来酰亚胺荧光团进行了特异性标记。每种蛋白都与16-S RNA形成复合物，当加入其他30-S核糖体蛋白时，形成了具有至少部分活性的完整30-S亚基。如果RNA的3'端也被标记（用荧光素），那么可以通过福斯特型能量转移来测量两个荧光团之间的距离。在核糖核蛋白复合物中，S4的结果是6.0纳米（60埃），在30-S亚基中是5.6纳米（56埃）；对于S17，在复合物中是6.3纳米（63埃），在亚基中是6.2纳米（62埃）。没有证据表明在30-S亚基形成过程中16-S RNA的3'端和5'端的相对位置发生了重大变化。讨论了误差来源，包括多重标记问题。为了更准确地测量能量转移程度，开发了一种基于酶消化的方法，并在本文中详细介绍。
