Créchet Jean-Bernard, Agbo'Saga Fulbert K, Baouz Soria, Hountondj Codjo
Ecole Polytechnique, Route de Saclay, F-91120 Palaiseau, France.
Sorbonne Université, Campus Pierre et Marie Curie, Unité de Recherche SUUR6 "Enzymologie de l'ARN", 7 Quai Saint-Bernard, F-75252 Paris Cedex 05, France.
Open Biochem J. 2018 Jul 31;12:113-129. doi: 10.2174/1874091X01812010113. eCollection 2018.
We have recently demonstrated that Lys-65 of the 62GANK65 motif of RpbL12 was affinity labeled with a tRNA analogue, resulting in the loss of activity.
In this report, we show that mutations operated at the position of Lys-65 led to an impairment in the activity of the mutant ribosomes, except the K65R or K65H bL12 mutants, suggesting that the only requirement of the reaction catalyzed or facilitated by RpbL12is the positive charge of the side chain of Lys-65. We also demonstrate that Lys-65 did not play any role in the peptidyl transferase reaction with respect to puromycin, but rather assisted the binding of the incoming aminoacyl-tRNA to the ribosomal A-site.
RESULTS & DISCUSSIONS: The protonated, positively charged εNH form of Lys-65 is likely to participate to the binding of aa-tRNA through ionic bonds with phosphate groups, in order to insure the accurate positioning required for the nucleophilic attack of its α-amino group on the carbonyl carbone of peptidyl-tRNA.
This α-NH group is likely to be generated by the unprotonated εNH form of Lys-65 which is capable of withdrawing a proton from the α-NH group of aa-tRNA.
我们最近证明，RpbL12的62GANK65基序中的赖氨酸65（Lys-65）被一种tRNA类似物进行了亲和标记，导致活性丧失。
在本报告中，我们表明，在赖氨酸65位置进行的突变导致突变核糖体活性受损，但K65R或K65H bL12突变体除外，这表明RpbL12催化或促进的反应的唯一要求是赖氨酸65侧链的正电荷。我们还证明，赖氨酸65在与嘌呤霉素的肽基转移酶反应中没有任何作用，而是协助进入的氨酰-tRNA与核糖体A位点结合。
赖氨酸65的质子化、带正电荷的εNH形式可能通过与磷酸基团的离子键参与氨酰-tRNA的结合，以确保其α-氨基对肽基-tRNA羰基碳进行亲核攻击所需的精确定位。
这个α-NH基团可能由赖氨酸65的未质子化εNH形式产生，该形式能够从氨酰-tRNA的α-NH基团中提取一个质子。
