Watson B S, Hazlett T L, Eccleston J F, Davis C, Jameson D M, Johnson A E
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Biochemistry. 1995 Jun 20;34(24):7904-12. doi: 10.1021/bi00024a015.
The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
利用荧光能量转移技术测定了氨酰 - tRNA·延伸因子Tu（EF - Tu）·GTP三元复合物中L形转运RNA的转角与结合GTP的EF - Tu之间的距离。供体染料荧光素（Fl）共价连接到苯丙氨酸tRNA（tRNAPhe）第8位的4 - 硫尿苷碱基上，氨酰化反应生成苯丙氨酰 - tRNAPhe - Fl8。GTP的核糖在2'（3'）位用受体染料罗丹明（Rh）进行共价修饰，形成GTP - Rh。通过EF - Tu对氨酰键化学水解的保护作用以及EF - Tu·GTP依赖的荧光素强度增加，验证了苯丙氨酰 - tRNAPhe - Fl8·EF - Tu·GTP - Rh三元复合物的形成。光谱分析表明，在苯丙氨酰 - tRNAPhe - Fl8·EF - Tu·GTP三元复合物中，荧光素的发射强度和寿命均大于苯丙氨酰 - tRNAPhe - Fl8·EF - Tu·GTP - Rh三元复合物。当在后者的三元复合物中加入过量GTP以取代GTP - Rh时，这些光谱差异消失，从而表明在三元复合物中激发态能量从荧光素转移到了罗丹明。单重态 - 单重态能量转移效率较低（10 - 12%），对应于三元复合物中供体和受体染料之间的距离为70±7 Å，其中所示的不确定性反映了染料取向的不确定性。校正探针连接链的长度后，GTP核糖的2'（3'） - 氧与s4U中的硫之间至少相隔49 Å。这个大距离限制了三元复合物中EF - Tu和tRNA可能的排列方式。（摘要截选至250字）
