Monitoring Translation with Modified MRNAs Strategically Labeled with Isomorphic Fluorescent Nucleosides

Abstract

To kinetically monitor translation-related events at a nucleotide resolution, a series of mRNAs, site-specifically modified at key codon positions with new emissive and responsive isomorphic nucleotides (thC, thU, thG - work withthA is commencing) have been studied. mRNAs modified with thC, thU and thG (excluding thU2 in the start codon) form an initiation complex (70SIC) and show significant spectral changes between the free and 70SIC-bound fluorescent mRNAs. All emissive mRNAs tested containing labeled nucleotides in the second codon facilitate aa-tRNA A-site binding [as part of an aa-tRNA.EF-Tu.GTP ternary complex (TC)] and pretranslocation (PRE) complex formation, although with somewhat different relative efficiencies, and all PRE complexes can be translocated via addition of EF-G.GTP to form postranslocation (POST) complexes. In most cases spectral differences are seen on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes, allowing measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Preliminary comparison of tRNA A-site binding kinetics, as monitored by the fluorescent mRNAs, with other measures of PRE complex formation indicate that TC binding to the ribosome is faster than codon-anticodon interaction, which in turn is somewhat faster than tRNA accommodation. These preliminary observations not only show excellent performance of mRNAs modified with our emissive RNA alphabet, but also clearly demonstrate their potential to illuminate the formation and disappearance of discrete intermediates in the polypeptide elongation cycle.