Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·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.

Non-catalytic regulation of gene expression by aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARSs) are a group of essential and ubiquitous "house-keeping" enzymes responsible for charging corresponding amino acids to their cognate transfer RNAs (tRNAs) and providing the correct substrates for high-fidelity protein synthesis. During the last three decades, wide-ranging biochemical and genetic studies have revealed non-catalytic regulatory functions of multiple AARSs in biological processes including gene transcription, mRNA translation, and mitochondrial RNA splicing, and in diverse species from bacteria through yeasts to vertebrates. Remarkably, ongoing exploration of non-canonical functions of AARSs has shown that they contribute importantly to control of inflammation, angiogenesis, immune response, and tumorigenesis, among other critical physiopathological processes. In this chapter we consider the non-canonical functions of AARSs in regulating gene expression by mechanisms not directly related to their enzymatic activities, namely, at the levels of mRNA production, processing, and translation. The scope of AARS-mediated gene regulation ranges from negative autoregulation of single AARS genes to gene-selective control, and ultimately to global gene regulation. Clearly, AARSs have evolved these auxiliary regulatory functions that optimize the survival and well-being of the organism, possibly with more complex regulatory mechanisms associated with more complex organisms. In the first section on transcriptional control, we introduce the roles of autoregulation by Escherichia coli AlaRS, transcriptional activation by human LysRS, and transcriptional inhibition by vertebrate SerRS. In the second section on translational control, we recapitulate the roles of GluProRS in translation repression at the initiation step, auto-inhibition of E. coli thrS mRNA translation by ThrRS, and global translational arrest by phosphorylated human MetRS. Finally, in the third section, we describe the RNA splicing activities of mitochondrial TyrRS and LeuRS in Neurospora and yeasts, respectively.

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.