Ermolenko Dmitri N, Spiegel P Clint, Majumdar Zigurts K, Hickerson Robyn P, Clegg Robert M, Noller Harry F
Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
Nat Struct Mol Biol. 2007 Jun;14(6):493-7. doi: 10.1038/nsmb1243. Epub 2007 May 21.
During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
在蛋白质合成过程中，转运RNA和信使RNA通过核糖体的A、P和E位点进行偶联转位，这一过程由延伸因子EF-G催化。紫霉素可阻断细菌核糖体上的转位过程，据信它结合在亚基界面处。利用荧光共振能量转移和化学足迹法，我们发现紫霉素将核糖体捕获在转位的中间状态。FRET效率的变化表明，紫霉素引起的两个核糖体亚基的相对移动与EF-G结合GDPNP所诱导的移动无法区分。化学探针实验表明，紫霉素诱导形成一种杂合状态的转位中间体。因此，紫霉素通过一种独特的机制抑制翻译，将核糖体锁定在杂合状态；冷冻电镜观察到的EF-G诱导的“棘轮”状态与杂合状态相同；而且，由于翻译对紫霉素敏感，杂合状态可能存在于体内。
