Andrei A. KorostelevCarbone, Christine E.2022-12-192022-12-192022-11-0710.13028/46qt-ke28https://hdl.handle.net/20.500.14038/51478Translation is the key step of gene expression whereby proteins are synthesized by the ribosome. This work addresses how translation factors EF-G and ArfB aid the ribosome in translation and mRNA quality control. Synchronous movement of mRNA and tRNAs through the ribosome is catalyzed by the bacterial GTPase Elongation Factor G (EF-G). EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive ribosome•EF-G intermediates at near-atomic resolution. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation. As the ribosome moves along the mRNA, it will stall if there is no stop codon in the A site due to mRNA damage or mutation. Such ribosome would be unable to terminate and require rescue. We asked how alternative rescue factor B (ArfB) rescues stalled ribosomes. We report that ArfB can recognize a wide range of mRNA substrates, including longer mRNAs extending beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB.en-USCopyright © 2022 CarboneAll Rights Reservedribosomecryo-EMtranslationbiochemistryDoctoral Dissertation0000-0001-5502-0859