Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

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• 作者：Wei Liu ; Darius Kavaliauskas ; Jared M. Schrader ; Kiran Poruri ; Victoria Birkedal ; Emanuel Goldman ; Hieronim Jakubowski ; Wlodek Mandecki ; Olke C. Uhlenbeck ; Charlotte R. Knudsen ; Yale E. Goldman ; Barry S. Cooperman
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：October 17, 2014
• 年：2014
• 卷：9
• 期：10
• 页码：2421-2431
• 全文大小：482K
• ISSN：1554-8937

文摘

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA路EF-Tu路GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.