Ribosome binding by tRNAs with fluorescent labeled 3' termini.
Ribosome binding by tRNAs with fluorescent labeled 3' termini.
复制标题
带有荧光标记 3 末端的 tRNA 与核糖体结合。
DOI：
10.1093/nar/8.14.3229
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发表时间：
1980
影响因子：
14.9
通讯作者：
Cantor,CR
中科院分区：
文献类型：
--
作者：
Wells,BD;Cantor,CR
酵母和大肠杆菌 RNAphe 样品被氧化并在 3' 端用 dansy1 肼或荧光素氨基硫脲标记。这些 tRNA 可以在 8°C 的 25 mM 镁中与聚（U）编程的大肠杆菌70S 紧密耦合核糖体结合。对于酵母 tRNAphe 衍生物，确定了两个结合常数约为 1 × 10°9 M−1 (P) 和 3×107M−1(A) 的结合位点，对于大肠杆菌，A 位点亲和力与酵母 tRNAphe 相似，但 P 位点亲和力弱 5 倍。单线态-单线态能量转移表明，tRNAphein 3′端的 P 位点到红霉素荧光素衍生物的距离为 23 Å。这支持了表明红霉素结合在肽基 tRNA 的肽部分附近的体外研究。还测量了同时结合在核糖体上的 2 个 tRNA 3' 端之间的 34 Å 距离。如此长的距离可能意味着脱酰化荧光 tRNA 以类似于严格响应中而不是蛋白质合成中的方向结合到 A 位点。
Yeast andE.colitRNAphesamples were oxidized and labeled at the 3′ end with dansy1 hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmedE.coli70S tight couple ribosomes in 25 mM magnesium at 8°C. Two binding sites with binding constants of about 1 × 10°9 M−1 (P) and 3×107M−1(A) were determined for the yeast tRNAphederivatives, WithE.colitRNAphethe A site affinity is similar to yeast tRNAphebut the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showed that the distance from the 3′ end of tRNAphein the P site to a fluorescein derivative of erythromycin is 23 Å. This supportsinvitrostudies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 Å between the 3′ ends of 2 tRNAs bound simultaneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.