Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
J Mol Biol. 2010 Mar 5;396(4):1043-52. doi: 10.1016/j.jmb.2009.12.043. Epub 2010 Jan 4.
EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis.
EF4 虽然在结构上与移位酶 EF-G 相似,但能促进核糖体上 tRNA 的反移位,在某些条件下对细菌生长很重要。在这里,我们使用了一系列协调的体外动力学测量方法,包括肽基-tRNA 的嘌呤霉素反应性和标记 tRNA 和 mRNA 的荧光变化,阐明了 EF4 催化反移位的动力学机制,并确定了移位抑制剂大观霉素和沃霉素对该过程的影响。EF4 依赖性反移位通过一个四步动力学方案从后移位(POST)复合物到前移位(PRE)复合物进行(即 POST-->I(1)-->I(2)-->I(3)-->PRE,这不是简单的移位逆转)。在反移位过程中,tRNA 核心区域和 mRNA 的运动紧密偶联,但有时与肽基-tRNA 3'末端的运动解偶联。EF4 可以被认为是反移位的中断催化,在形成 I(3)时停止,而不是催化反移位的完整过程,最终形成 PRE 复合物。由于 I(3)的瞬时积累导致多肽延伸的延迟,这可能对优化功能性蛋白质生物合成很重要。