Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with –1 frameshifting.

Ribosomal frameshifting is an important pathway used by many viruses for protein synthesis that involves mRNA translocation of various numbers of nucleotides. Resolving the mRNA positions with subnucleotide precision will provide critical mechanistic information that is difficult to obtain with current techniques. We report a method of high‐resolution DNA rulers with subnucleotide precision and the discovery of new frameshifting intermediate states on mRNA containing a GA 7 G motif. Two intermediate states were observed with the aid of fusidic acid, one at the “0” reading frame and the other near the “−1” reading frame, in contrast to the “−2” and “−1” frameshifting products found in the absence of the antibiotic. We termed the new near‐“−1” intermediate the Post(−1*) state because it was shifted by approximately half a nucleotide compared to the normal “−1” reading frame at the 5’‐end. This indicates a ribosome conformation that is different from the conventional model of three reading frames. Our work reveals uniquely precise mRNA motions and subtle conformational changes that will complement structural and fluorescence studies.