GTP酶
EF图
GTP'
突变体
核糖体
生物
突变
小型GTPase
鸟苷三磷酸
翻译(生物学)
大肠杆菌
生物化学
细胞生物学
化学
生物物理学
信号转导
酶
核糖核酸
基因
信使核糖核酸
作者
Barend Kraal,Leo Zeef,J.R. Mesters,Kathy Boon,Erik L.H. Vorstenbosch,L. Bosch,Pieter H. Anborgh,Andrea Parmeggiani,Rolf Hilgenfeld
摘要
Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kir r ) or pulvomycin (pul r ). All kir r mutations cluster in the interface of domains 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu∙GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu∙GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pul r mutations cluster in the three-domain junction interface of EF-Tu∙GTP (which is an open hole in EF-Tu∙GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99–R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. Kir r EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.Key words: kirromycin, pulvomycin, streptomycin, GTPase switch, aminoacyl-tRNA.
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