Rychlik W, Odom O W, Hardesty B
Biochemistry. 1983 Jan 4;22(1):85-93. doi: 10.1021/bi00270a012.
Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.
荧光技术被用于研究肽链延长因子Tu（EF-Tu）与大肠杆菌核糖体的结合，并确定结合因子与核糖体上各点之间的距离。嗜热栖热菌EF-Tu用3-（4-马来酰亚胺基苯基）-4-甲基-7-（二乙氨基）香豆素（CPM）标记，活性未丧失。在苯丙氨酰-tRNA和一种不可水解的GTP类似物存在的情况下，70S核糖体结合CPM-EF-Tu [结合常数Kb =（3±1.2）×10⁶ M⁻¹]，导致CPM荧光减弱。CPM-EF-Tu与50S亚基的结合比与70S核糖体的结合至少低一个数量级，且未检测到与30S亚基的结合。含有用荧光素马来酰亚胺标记的S1或用荧光素硫代半卡巴腙在其3'末端标记的核糖体RNA的重组70S核糖体用于CPM-EF-Tu的能量转移。结合到核糖体上的CPM-EF-Tu与16S RNA、5S RNA、23S RNA的3'末端以及S1最接近的巯基之间的距离分别计算为82、70、73和62 - 68埃。
