Investigating Ribosomal Translocation Mechanism with Precise Magnetic DNA Rulers

This dissertation focuses on developing precise magnetic DNA rulers to investigate ribosomal translocation mechanism and measure the power stroke of translocase EF-G. The precise 3-nucleotide movement of mRNA is critical for translation fidelity. One mRNA translocation error propagates to all of the following codons, which is detrimental to the cell. However, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. Through magnetic labeling on the 3ʹ-ends of the mRNA, we observed efficient “-1” and “-2” frameshiftings on a mRNA containing a GA7G slippery motif without the downstream secondary structure. The detection technique we used was force-induced remnant magnetization spectroscopy (FIRMS), which was invented by our group. The result represented the first experimental evidence of multiple frameshifting steps. To further reveal the mRNA dynamics near the ribosome entry site, I have developed an assay of dual magnetic DNA rulers that uniquely probe both the 3ʹ- and 5ʹ-ends of mRNA. An antibiotic-trapped intermediate state was observed, which indicated a novel ribosomal conformation containing mRNA asymmetric partial displacements at its entry and exit sites. Based on the available ribosome structures and computational simulations, we proposed a “looped” mRNA conformation, which suggested a stepwise “inchworm” mechanism for ribosomal translocation. The same “looped” intermediate state identified with the dual rulers persists with a “-1” frameshifting motif, indicating that the branching point of normal and frameshifting translocations occurs at a later stage of translocation. In the last, we reported quantitative measurements of the power strokes of structurally modified EF-Gs using both magnetic and microscope detections. The results showed that the power stroke was reduced by 30 pN when the EF-G was restrained by a short crosslinking molecule or by the binding of fucidic acid. The results also showed that the reduced power stroke only lowered the percentage of translocation but did not introduce translocation error. Furthermore, the microscope detection method that I developed produced consistent results with the magnetic detection using FIRMS. Compared to magnetic detection, microscope detection is more straightforward, cheaper, and easier to implement. Therefore, it may be adapted to measuring other forces in biological systems.