Ganoza M C, Zahid N, Baxter R M
Eur J Biochem. 1985 Jan 15;146(2):287-94. doi: 10.1111/j.1432-1033.1985.tb08651.x.
The requirements for peptide-bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet-tRNA as donor substrate and puromycin or alpha-hydroxypuromycin as acceptors. We report that the soluble protein EF-P, purified to apparent homogeneity, stimulates the synthesis of N-formylmethionylpuromycin or N-formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits. In the presence of EF-P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide-bond synthesis or transesterification. The involvement of 50S subunit proteins in EF-P-stimulated peptide-bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and 'split' proteins, several of which were purified to homogeneity. When added to 30S X A-U-G X f[35S]Met-tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide-bond synthesis or transesterification poorly. EF-P stimulated peptide-bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF-P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide-bond-forming and transesterification activities and EF-P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF-P-stimulated peptide-bond and transesterification reactions of the peptidyl transferase. Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide-bond formation and transesterification. Similarly ethoxyformylation of EF-P resulted in a 25-50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF-P in the catalytic mechanism of peptidyltransferase is discussed.
以甲硫氨酰 - tRNA作为供体底物，嘌呤霉素或α - 羟基嘌呤霉素作为受体，研究了大肠杆菌70S核糖体、50S天然或重构50S亚基的肽键合成和转酯反应的要求。我们报道，纯化至表观均一的可溶性蛋白EF - P可刺激70S核糖体或重新结合的30S和50S亚基合成N - 甲酰甲硫氨酰嘌呤霉素或N - 甲酰甲硫氨酰羟基嘌呤霉素。在EF - P存在下，70S核糖体在催化肽键合成或转酯反应方面比50S颗粒效率显著更高。研究了50S亚基蛋白在EF - P刺激的肽键形成和转酯反应中的作用。50S亚基被2.0 M LiCl解离为核心颗粒和“裂解”蛋白，其中几种被纯化至均一。当添加到30S X A - U - G X f[35S]Met - tRNA时，50S核心颗粒或用L6或L11重构的50S核心颗粒促进肽键合成或转酯反应的能力较差。EF - P对这两种类型的核心颗粒刺激肽键合成的程度大致相同。另一方面，EF - P刺激用L6和L11重构的核心颗粒发生低水平的转酯反应。相比之下，用L16重构的核心颗粒表现出肽键形成和转酯活性，EF - P分别将这两种反应刺激了20倍和40倍。因此，不同的蛋白质对肽基转移酶的内在或EF - P刺激的肽键和转酯反应有不同的刺激作用。用于重构核心颗粒的50S亚基或纯化的L16进行乙氧甲酰化后，导致肽键形成和转酯反应丧失。同样，EF - P的乙氧甲酰化导致其刺激这两种反应的能力丧失25 - 50%。30S亚基对该试剂处理有抗性。这些结果表明组氨酸残基参与肽基转移酶活性。讨论了EF - P在肽基转移酶催化机制中的作用。
