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 3′ 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 3′ end. Using Escherichia coli tRNA^Cys 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 high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNA^Cys 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 PyC-labeled 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.

Keywords

• pyrrolo-cytosine ribonucleoside
• aminoacylation
• ribosome translocation
• tRNACys
• tRNA CCA adding enzyme
• fluorescence