Dual DNA rulers reveal an ‘mRNA looping’ intermediate state during ribosome translocation

Abstract

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, none of the current methods can reveal the mRNA dynamics near the ribosome entry site, which limits the understanding of this important issue. We have developed an assay of dual DNA rulers that provides such capability. By uniquely probing both the 3ʹ- and 5ʹ-ends of mRNA, we observed an antibiotic-trapped intermediate state that is consistent with a 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.

Modulation and Visualization of EF‐G Power Stroke During Ribosomal Translocation

“…The 3 bp difference was caused by the normal translocation step in which three more nucleotides on the mRNA would be covered by the ribosome in the Post and could no longer hybridize with the probing DNA (Figure A, bottom). If frameshifting occurred, we would observe a 13 bp duplex for −1 frameshifting, or a 14 bp duplex for −2 frameshifting, as we have demonstrated in previous publications . The results of the ribosome positions and their corresponding percentages are shown in Figure B.…”

“…We investigated the effect of reduced power strokes on translocation by identifying the exact ribosome movement on the mRNA. The probing scheme is shown in Figure A, which has been used in our previous publications . Briefly, the ribosome position was revealed by the number of bp between the exposed mRNA and the probing DNA; the number of bp was deduced from the critical force of the duplexes obtained by FIRMS.…”

High‐Resolution DNA Dual‐Rulers Reveal a New Intermediate State in Ribosomal Frameshifting

“…The 5'-end DNA rulers contained a 50-nt linker to overcome the steric hindrance between the surface and the ribosome. [13] The precise lengths of the mRNA uncovered by the ribosome were determined by the dissociation forces of the DNA-mRNA duplexes. Therefore, the ribosome footprints will be deduced.…”

“…The correlation of the duplex dissociation force and the ribosome's coverage on the mRNA has been demonstrated earlier, in which we found that the ribosome covers 27 nt in the pre-and post-states, while the first mRNA nt out of the leading edge (3'-end) is + 12 from the first nucleotide in the P-site. [13,18] Force spectra of the duplexes were obtained by measuring the magnetic signal of the sample as a function of acoustic radiation force exerted on the duplexes via the magnetic bead, in which the dissociation force was indicated by a decrease of the magnetic signal. Figure 1b shows the force spectra of DNA rulers that formed 12-bp duplex with the 5'-end mRNA, in the presence and absence of the ribosome in the pre-translocation state (Pre).…”

“…We recently developed DNA rulers based on force-induced remnant magnetization spectroscopy (FIRMS). [13] With 2-4 pN force resolution, we were able to probe the mRNA positions with single-nt resolution and observed a looped mRNA conformation trapped by antibiotics. Meanwhile on the technical front, we invented super-resolution force spectroscopy (SURFS) by implementing acoustic radiation force, which was automatable and more precise than the centrifugal force used in FIRMS.…”