Hountondji Codjo, Créchet Jean-Bernard, Le Caër Jean-Pierre, Lancelot Véronique, Cognet Jean A H, Baouz Soria
Sorbonne Universités UPMC Univ Paris 06, Unité de Recherche UPMC UR6 "Enzymologie de l'ARN", 4, Place Jussieu, F-75252 Paris Cedex 05, France.
Ecole Polytechnique, Route de Saclay, F-91120 Palaiseau, France.
J Biochem. 2017 Dec 1;162(6):437-448. doi: 10.1093/jb/mvx055.
In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E. coli bL12 and human eL42 proteins display significant primary structure similarities, which argues for evolutionary conservation of these two proteins located at the tRNA-CCA binding site on eubacterial and eukaryal ribosomes.
在本报告中，我们使用了高碘酸盐氧化的tRNA（tRNAox）作为亲和标记试剂，以证明：（i）bL12蛋白与大肠杆菌70S核糖体上P位点结合的tRNA的CCA臂接触；（ii）标记的化学计量是一分子tRNAox与内源性bL12的一条多肽链结合；（iii）核糖体上bL12与tRNAox的原位交联会导致活性丧失；（iv）完整的tRNA可保护70S核糖体中的bL12不与tRNAox交联；（v）tRNAox和磷酸吡哆醛（PLP）竞争核糖体内部bL12上相同或近端的交联位点；（vi）PLP与重组大肠杆菌bL12蛋白交联的化学计量是每条多肽链共价结合一分子PLP；（vii）与PLP交联的重组bL12的氨基酸残基是Lys-65；（viii）大肠杆菌bL12的Lys-65对应于eL42的Lys-53，先前已证明eL42在原位可与人80S核糖体上P位点结合的tRNAox交联；（ix）最后，大肠杆菌bL12和人eL42蛋白显示出显著的一级结构相似性，这表明位于真细菌和真核生物核糖体上tRNA-CCA结合位点的这两种蛋白在进化上具有保守性。
