Aoki H, Adams S L, Turner M A, Ganoza M C
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Biochimie. 1997;79(1):7-11. doi: 10.1016/s0300-9084(97)87619-5.
The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced. We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we report the purification, crystallization and initial characterization of the efp gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin. This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.
翻译因子EF-P是在70S核糖体上从甲酰甲硫氨酸-tRNAfMet高效合成原核生物肽键所必需的。该蛋白已从大肠杆菌细胞中纯化出来，编码它的基因efp也已被克隆和测序。我们分离出了重组克隆，这些克隆在SDS-PAGE分析中过表达一种与纯化的EF-P共迁移的蛋白。利用这些克隆，我们报道了efp基因产物的纯化、结晶及初步表征。使用几种抑制转位、肽键合成和解码的抗生素研究了EF-P刺激肽键合成的机制。EF-P对肽基转移酶的刺激不受影响转位和A位点占据（在延伸状态下）的抗生素（即硫链丝菌素、紫霉素、新霉素和夫西地酸）的抑制，但受链霉素以及肽基转移酶抑制剂氯霉素和林可霉素的抑制。这一观察结果以及对L16而非L7/L12、L6或L11核糖体蛋白的需求表明，EF-P的结合位点可能与核糖体的肽基转移酶中心重叠。
