Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells. In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins. This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site. Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift. The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function.