During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome’s control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.

Elongation factor G (EF-G) is a translational GTPase that acts at several stages of protein synthesis. Its canonical function is to catalyze tRNA movement during translation elongation, but it also acts at the last step of translation to promote ribosome recycling. Moreover, EF-G has additional functions, such as helping the ribosome to maintain the mRNA reading frame or to slide over non-coding stretches of the mRNA. EF-G has an unconventional GTPase cycle that couples the energy of GTP hydrolysis to movement. EF-G facilitates movement in the GDP-Pi form. To convert the energy of hydrolysis to movement, it requires various ligands in the A site, such as a tRNA in translocation, an mRNA secondary structure element in ribosome sliding, or ribosome recycling factor in post-termination complex disassembly. The ligand defines the direction and timing of EF-G-facilitated motion. In this review, we summarize recent advances in understanding the mechanism of EF-G action as a remarkable force-generating GTPase.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP—Pi form stabilizes ribosomal subunit rotation and induces twisting of the sarcin-ricin loop of the 23S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a cascade of rearrangements and a large-scale rotation of EF-G that exerts force on the ribosome and ultimately drives tRNA movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.