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. Author manuscript; available in PMC: 2023 Apr 30.
Published in final edited form as: J Mol Biol. 2022 Jan 4;434(8):167440. doi: 10.1016/j.jmb.2021.167440

Figure 6.

Figure 6.

Ensemble kinetic assays to monitor +1 frameshifting of SufB2 in the P site. (A) A POST complex harboring the dipeptidyl-ProL in the P site is formed on the mRNA sequence AUG-CCC-CGU-U. The POST complex is stalled over time and any change of the reading frame in the P site is monitored by addition of an equimolar mixture of Val-TC and Arg-TC. The result identifies synthesis of fMPV, indicating evidence of +1 frameshifting of the POST complex. (B) A 70SIC harboring fMet-tRNAfMet in the P site, programmed with the same mRNA as above, is rapidly delivered with an equimolar mixture of SufB2-TC and Val-TC or with an equal mixture of SufB2-TC and Arg-TC during active protein synthesis. The result reveals complete synthesis of fMPV when the 70SIC is rapidly mixed with SufB2-TC and Val-TC, but complete synthesis of fMPR when the 70SIC is rapidly mixed with SufB2-TC and Arg-TC, providing evidence of frameshifting in the P site depending on the reading-frame occupancy of the A-site tRNA.