Hou Y, Yaskowiak E S, March P E
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
J Bacteriol. 1994 Nov;176(22):7038-44. doi: 10.1128/jb.176.22.7038-7044.1994.
The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.
核糖体在mRNA上的转位是由细胞机制完成的，该机制在整个生物物种范围内都得到了极其良好的保守。这个过程需要延伸因子G（EF-G，在古细菌和真核生物中为EF-2），它是GTPase超家族的成员。利用遗传技术，我们鉴定出了fusA（编码大肠杆菌EF-G的基因）的一系列突变等位基因，这些等位基因在体内无法支持蛋白质合成。这些等位基因编码的蛋白质在密码子495（密码子495处Q-to-P变化的变体[Q495P]）、502（G502D）和563（G563D）处有单点突变，在密码子608处有一个无义突变。生化分析表明，EF-G Q495P、G502D和delta 608-703在鸟嘌呤核苷酸结合方面没有受到破坏，但在核糖体依赖性GTP水解和鸟嘌呤核苷酸依赖性核糖体结合方面存在缺陷。我们提出，所有这些突变都存在于一个对核糖体结合至关重要的结构域中，并且GTP水解缺陷是与70S核糖体结合受损的次要后果。
