Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Abstract

All mature tRNA molecules have the conserved CCA sequence at the 39 end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 39 end. Using Escherichia coli tRNACys as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA Cys , separated from the unlabeled precursor tRNA by reverse phase highpressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNACys with E. coli CysRS reveals an equilibrium K d closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyClabeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

Direct Evaluation of tRNA Aminoacylation Status by the T-Box Riboswitch Using tRNA-mRNA Stacking and Steric Readout

“…To probe the structural context of C178 in the complex, we employed the fluorescent cytosine analog pyrrolocytosine (PyC). PyC forms a base pair with G that exhibits structural and thermodynamic properties of a normal G•C pair, and its fluorescence is sensitive to its environment (Buskiewicz and Burke, 2012; Tinsley and Walter, 2006; Zhang et al, 2008; Zhang and Wadkins, 2009). To introduce PyC at T-box position 178, we nicked the non-conserved loop of the antiterminator between positions 170 and 171, generating a two-piece variant of T-box 182 .…”

A Flexible, Scalable Method for Preparation of Homogeneous Aminoacylated tRNAs

“…For tRNAs esterified with aromatic or other highly hydrophobic side chains, aa-tRNAs and non-aa-tRNAs can be directly separated using RP-HPLC, based on their difference in hydrophobicity (Cayama, Yepez, Rotondo, Bandeira, Ferreras et al, 2000; Zhang, Liu, Christian, Gamper, Rozenski et al, 2008). For tRNAs esterified with other amino acids, in particular those with small or polar side chains (e.g., glycine, alanine), chromatographic methods are generally unable to achieve satisfactory separation.…”

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

“…The penultimate base of the appropriate tRNALeu (UAA) isoacceptor was labelled with a pyrrolo-cytidine fluorophore. To achieve labelling, E. coli tRNA nucleotidyl transferase enzyme was incubated with a truncated in vitro tRNA transcript and pyrrolo-CTP (Trilink Biotechnologies) and ATP as outlined previously 32 . Fluorescence experiments were performed in buffer containing 50 mM HEPES (pH 7.4), 20 mM KCl, 10 mM MgCl 2 and 10 mM b-ME at 28 1C.…”

“…3c). This approach was used to monitor the conformation of the CCA 3 0 -end of tRNA Cys interacting with cysteinyl-tRNA synthetase 32 . By monitoring the fluorescence from this probe, we expected to see a significant change in the spectrum when the LeuRS Á tRNApyr Leu complex was bound to TM84 or not.…”