Monitoring Translation with Modified mRNAs Strategically Labeled with Isomorphic Fluorescent Guanosine Mimetics

Here we examine three mRNAs, site-specifically modified at codon positions 4, 5, and 6 with a new emissive and responsive isosteric guanosine mimetic ((th)G), with the goal of developing real time assays for monitoring translation-related events at nucleotide resolution. All three emissive mRNAs tested form initiation (70SIC), pretranslocation (PRE), and posttranslocation (POST) complexes. In most cases spectral differences are seen on binding of the mRNA to the ribosome during 70SIC formation and on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes. These differences allow measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Such measurements directly identify a specific step during PRE complex formation, provisionally assigned to codon:anticodon-loop base pair formation, that follows aa-tRNA.EF-Tu.GTP ternary complex binding to the ribosome and precedes aa-tRNA accommodation into the A-site of the ribosome. These observations demonstrate not only the functionality of mRNAs modified with the emissive guanosine mimetic but also the potential this mimetic offers for observing the formation and disappearance of discrete intermediates during the polypeptide elongation cycle.