Odom O W, Robbins D J, Lynch J, Dottavio-Martin D, Kramer G, Hardesty B
Biochemistry. 1980 Dec 23;19(26):5947-54. doi: 10.1021/bi00567a001.
The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.
从大肠杆菌核糖体中分离出三种核糖核酸（RNA），然后通过高碘酸盐氧化，接着与荧光素或曙红的硫代氨基脲反应，在其3'端进行标记。用标记的RNA重建的核糖体亚基对多聚苯丙氨酸合成具有活性。通过从荧光素到曙红的非辐射能量转移来估计70S核糖体中RNA的3'端之间的距离。能量转移的百分比是根据淬灭样品中荧光素的荧光寿命相对于未淬灭样品的降低来计算的。通过使用锁模激光进行激发和高速静电光电倍增管进行荧光检测来实时测量荧光寿命。连接到16S RNA和5S RNA或23S RNA的3'端的荧光团之间的距离分别估计约为55和71埃。5S RNA和23S RNA之间的相应距离太大，无法用现有的探针可靠测量，但估计大于65埃。对溶液中游离的和重建到核糖体亚基中的标记RNA的量子产率进行比较表明，16S RNA的3'端与其他核糖体成分没有明显相互作用，可能处于相对暴露的位置，而5S RNA和23S RNA的3'端可能埋在70S核糖体亚基中。
