DISTANCE MOVED BY TRANSFER-RNA DURING TRANSLOCATION FROM THE A SITE TO THE P SITE ON THE RIBOSOME
DISTANCE MOVED BY TRANSFER-RNA DURING TRANSLOCATION FROM THE A SITE TO THE P SITE ON THE RIBOSOME
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DOI：
10.1016/0022-2836(82)90462-4
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发表时间：
1982-01-01
影响因子：
5.6
通讯作者：
CANTOR, CR
中科院分区：
文献类型：
--
作者：
JOHNSON, AE;ADKINS, HJ;CANTOR, CR
单重态-单重态能量转移用于测量与同一核糖体结合的2个转移RNA分子的中段之间的距离。通过将荧光染料共价连接到4-硫代尿苷上来修饰两种大肠杆菌tRNA：tRNAfMet与5-[2-（2-碘乙酰胺基）乙氨基]-1-萘磺酸（IAEDANS）反应得到tRNAfMet-AEDANS 8，tRNAPhe与5-碘乙酰胺基荧光素反应得到tRNAPhe-F8。这些荧光标记的tRNA通过RPC-5色谱法纯化。修饰的tRNAfMet-AEDANS 8与未修饰的tRNA与甲硫氨酰-tRNA合成酶、转化酶、起始因子、核糖体和肽基转移酶的相互作用达到相同的程度。tRNAPhe-F8与苯丙氨酰-tRNA合成酶、延伸因子EF-Tu相互作用的能力。GTP复合物和核糖体复合物的荧光染料的存在下，只有轻微减少。通过使用A-U-G-U4-5信使将fMet-tRNAfMet-AEDANS 8（供体）结合到P位点，然后使用EF-Tu. cnt. dot将Phe-tRNAPhe-F8（受体）结合到A位点，制备用于能量转移实验的核糖体复合物。GTP使用未修饰的tRNA代替供体、受体或两者平行制备必要的对照样品。供体染料在P位点的荧光被强烈猝灭（约70%）。这种高效率的能量转移对应于26 ± 100 nm的分离距离。4)ANG。在染料之间。因此，结合在A和P位点的tRNA的中段彼此靠近，仅相隔2-10埃，从A位点到紧邻的P位点的易位需要< 30埃的移动。tRNA的这种排列表明，脱酰化的tRNA从P位点的排出可能是由结合在A和P位点的tRNA之间的静电排斥提供动力的。
Singlet-singlet energy transfer was used to measure the distance between the midsections of 2 transfer RNA molecules bound to the same ribosome. Two Escherichia coli tRNA species were modified by the covalent attachment of a fluorescent dye to 4-thiouridine: tRNAfMet was reacted with 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid (IAEDANS) to give tRNAfMet-AEDANS8, and tRNAPhe with 5-iodoacetamidofluorescein to give tRNAPhe-F8. These fluorescent-labeled tRNA were purified by chromatography on RPC-5. The modified tRNAfMet-AEDANS8 interacted to the same extent as the unmodified tRNA with methionyl-tRNA synthetase, transformylase, initiation factors, ribosomes and the peptidyltransferase. The ability of tRNAPhe-F8 to interact with phenylalanyl-tRNA synthetase, elongation factor EF-Tu .cntdot. GTP complex and the ribosomal complex was only slightly diminished by the presence of the fluorescein dye. Ribosomal complexes were prepared for the energy transfer experiments by binding fMet-tRNAfMet-AEDANS8 (the donor) to the P site using an A-U-G-U4-5 message, and then binding Phe-tRNAPhe-F8 (the acceptor) to the A site using EF-Tu .cntdot. GTP. The necessary control samples were prepared in parallel using unmodified tRNA in place of the donor, the acceptor or both. The fluorescence of the donor dye in the P site was strongly quenched (.apprx. 70%) when the acceptor dye was present in the A site. This high efficiency of energy transfer corresponds to a separation distance of 26(.+-. 4) .ANG. between the dyes. The midsections of the tRNA bound in the A and P sites are therefore located close to each other, separated by only 2-10 .ANG., and translocation from the A site into the immediately adjacent P site requires a movement of < 30 .ANG.. This arrangement of the tRNA suggests that the ejection of the deacylated tRNA from the P site might be powered by the electrostatic repulsion between the tRNA bound in the A and P sites.