Abstract
The C5 methylation of cytidine 1407 (C1407) in the E. coli 16S rRNA is a permanent modification. The methylation occurs directly at the A-site internal loop, a known dynamic hotspot in helix 44 (H44) that undergoes conformational exchange between a functional ground and an excited state. Here, we show by relaxation dispersion NMR spectroscopy that methylation of C1407 reshapes the folding kinetics of the A-site RNA and prolongs the lifetimes of its ground and excited states. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments of unmodified and m(5)C1407-modified A-site RNA reveal that C5 methylation slows down the transition rate between the ground and excited states by 1.6-fold. This kinetic effect increases the lifetimes of the excited and ground states, with the latter playing a critical role in the correct codon anti-codon recognition during the elongation phase of translation. Thus, C5 methylation of C1407 functions as a kinetic regulator in the E. coli rRNA to properly adjust the time frame for the selection of the correct tRNA-mRNA interaction.