N (6)-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

We used quench flow to study how N(6)-methylated adenosines (m(6)A) affect the accuracy ratio between k(cat)/K(m) (i.e. association rate constant (k(a)) times probability (P(p)) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated k(cat)/K(m) for Glu-tRNA(Glu), EF-Tu and GTP forming ternary complex (T(3)) reading cognate (GAA and Gm(6)AA) or near-cognate (GAU and Gm(6)AU) codons. k(a) decreased 10-fold by m(6)A introduction in cognate and near-cognate cases alike, while P(p) for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated k(cat)/K(m) for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um(6)AA) and near-cognate (UAG and Um(6)AG) stop codons to decrease 6-fold or 3-fold by m(6)A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m(6)A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate k(cat)/K(m), in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate k(cat)/K(m).