Allosteric Effects of EF-G Domain I Mutations Inducing Ribosome Frameshifting Revealed by Multiplexed Force Spectroscopy

Abstract

Ribosome translocation catalyzed by elongation factor G (EF-G) is a critical step in protein synthesis where the ribosome typically moves along the mRNA by three nucleotides at each step. To investigate the mechanism of EF-G catalysis, it is essential to precisely resolve the ribosome motion at both ends of the mRNA, which, to our best knowledge, is only achieved with the magnetic-based force spectroscopy developed by our groups. Here, we introduce a novel multiplexed force spectroscopy technique that, for the first time, offers single-nucleotide resolution for multiple samples. This technique combines multiple acoustic force generators with the smallest atomic magnetometer designed for biological research. Utilizing this technique, we demonstrate that mutating EF-G at the GTP binding pocket results in the ribosome moving only two nucleotides on both ends of the mRNA, thereby compromising ribosome translocation. This finding suggests a direct link between GTP hydrolysis and ribosome translocation. Our results not only provide mechanistic insights into the role of GTP binding pocket but also illuminate how allosteric mutations can manipulate translocation. We anticipate broader applications of our technique in the ribosome field, leveraging its high efficiency and single-nucleotide resolution.

Keywords: Allosteric effect; DNA ruler; Elongation factor G mutation; Frameshifting; Multiplexed force spectroscopy; Ribosome translocation.

Figure 1.

Structure of the ribosome complex trapped at a translocation intermediate state (7pjv). The ribosome’s large (50S) and small subunits (30S) are shown as surface. The P-site tRNA and EF-G that occupies the A-site are shown in blue and domain-colored, respectively. The A- and E-site tRNAs are not shown for display clarity. The GTP binding pocket is highlighted in the red circle and enlarged at the right. The mutated residue T48 is colored red at the right. The Pi molecule after GTP hydrolysis but before Pi releasing is colored orange. The EF-G is flipped at the right for display clarity.

Figure 2.

Multiplexed force spectroscopy. (A) Schematic. 1: atomic sensor; 2: sample holder with five samples mounted on a linear motor; 3: five piezo disks in parallel; 4: water layer. (B) Photo of the atomic sensor. (C) Photo of the sample holder and acoustic force generation.

Figure 3.

Magnetic sensitivity and multiplexed capability of the new instrument for ribosome study. (A) Sensitivity of the atomic magnetometer. (B) Construct of the sample system. (C) Resolving five samples in a single detection scan.

Figure 4.

Force calibration for the new instrument. (A-E) Force spectra of 11–15 bp DNA duplexes, respectively. (F) Force calibration.

Figure 5.

Conformations of the GTP binding pocket and Pi release. (A, B) Surface representation of the GTP binding pocket for the closed (A, 7pjv) and open (B, 7pjy) conformations, respectively. (C) Enlarged surface representation of the switch I loop in the closed and open forms. To release Pi after GTP hydrolysis, the switch I loop needs to flip downward, as indicated by the arrow, to generate the escaping channel.

Figure 6.

Ribosome motion at the 3′-end of the mRNA. (A) Schemes of duplex formation between the exposed mRNA in the Pre complex and various DNA probes. (B) Force spectra. Identical dissociation forces at 49 pN were revealed for both P14 and P14b (the overlapping black and blue traces), whereas a lower force of 38 pN was observed using P13 (the red trace). (C) Probing scheme using P14 DNA for both the Pre and Post complexes. (D) Force spectra for the three EF-Gs. (E) Probing scheme using P11 and P12 DNAs for the Pre complex. (F) Force spectra using the shorter DNAs. The force axis was plotted in descending order to be consistent with the left to right translocation direction depicted in the schemes, which would produce shorter duplexes at the 3′-end.

Figure 7.

Ribosome motion at the 5′-end of the mRNA. (A) Probing scheme using P15_5′ DNA for both the Pre and Post complexes. (B) Force spectra for the three EF-Gs probed by P15_5′. (C) Probing scheme using P14_5′. The red arrow indicates the one less nt compared to P15_5′. (D) Force spectrum for translocation under EF-G WT probed by the shorter P14_5′. The force axis was plotted in ascending order to be consistent with the left to right translocation direction depicted in the schemes, which would produce longer duplexes at the 5′-end.

Figure 8.

Comparison of the relative reactivities of the three EF-Gs. Both 5′- and 3′-end translocations are measured for each EF-G.

Figure 9.

Proposed model for the frameshifting caused by mutated EF-Gs of T48E and T48V.