Biology subjects

Loveland, A. B.

Publications and source records attributed to Loveland, A. B..

7 recordsLinked to original sources

bioRxiv2025

In extracto cryo-EM reveals eEF2 as a major hibernation factor on 60S and 80S particles

Cryogenic electron microscopy (cryo-EM) made impressive progress in resolving cellular macromolecules and their detailed interactions. Single-particle cryo-EM traditionally relies on purified macromolecules and lacks the complexity of cellular environments, whereas in situ cryo-EM or cryo-ET require extensive sample preparation and data acquisition, presenting challenges in achieving high resolution. We describe cryo-EM of cellular lysates--in extracto cryo-EM--allowing the flexibility and high-resolution of cryo-EM in the context of cellular components. High-resolution 2D template matching (2DTM) yields [~]2.2 [A] maps of the mammalian translational apparatus. Elongating ribosome abundances in primate cell lines (MCF-7 and BSC-1) and rabbit reticulocyte lysates range from [~]70% to [~]10%, reflecting translational stress responses. Non-translating (hibernating) ribosomes carrying no mRNA, feature numerous proteins shielding ribosomal functional centers. Elongation factor 2 (eEF2) is the most abundant hibernation factor bound to >95% of ribosomes and, unexpectedly, to 60S subunits. eEF2*GDP is stabilized by interactions with the sarcin-ricin loop and protein uL14. Hibernating ribosomes also feature LARP1 involved in initiation and mTOR signaling; eIF5A implicated in elongation and termination; and other factors, exposing the variety of hibernation scenarios. Our work underscores the efficiency and potential of in extracto cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.

molecular biology↗

bioRxiv2025

Structural mechanism of mRNA decoding by mammalian GTPase GTPBP1

GTP-binding protein 1 (GTPBP1) is a widespread translational GTPase closely related to elongation factor eEF1A. The loss of GTPBP1 leads to errors in neuronal development in animals and is associated with neurodegenerative disorders in humans. Although linked to translation and quality control mechanisms, GTPBP1 functions remain largely obscure. Similarly to eEF1A, GTPBP1 delivers cognate aminoacyl-tRNA to the ribosomal A site in a GTP-dependent manner, but GTP hydrolysis is not followed by rapid peptide bond formation, and GTPBP1-mediated elongation is slow. To establish the basis for GTPBP1 function, we determined cryo-EM structures of 80S ribosomal complexes bound to GTPBP1*aa-tRNA with GTP or the non-hydrolysable analog GDPCP. They revealed that the unique GTPBP1 architecture, including the additional eIF1/IF3-like N-terminal domain and the shoulder-interacting H-loop in place of the 2 helix of canonical GTPases, is responsible for establishing GTPBP1-specific interactions with tRNA and the ribosome, leading to slow GTPBP1 dissociation after GTP hydrolysis and thus delayed tRNA accommodation. Slow dissociation correlates with an extended proofreading stage resulting in higher accuracy of GTPBP1-mediated decoding and potentially allows GTPBP1 to elicit its putative quality control functions. GTPBP1 visualization provides the foundation for mapping and elucidating GTPBP1 mutations associated with human diseases.

biochemistry↗

bioRxiv2023

Structural mechanism of angiogenin activation by the ribosome

Angiogenin, an RNase A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases, and epigenetic inheritance. Upon activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, producing nicked tRNA (also known as tRNA halves, tiRNAs or tsRNAs) and resulting in translation repression. The catalytic activity of isolated angiogenin, however, is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle. Here, we uncover these mechanisms using biochemical assays and cryogenic electron microscopy. Our work reveals that the cytosolic ribosome is the long-sought activator of angiogenin. A 2.8-[A] resolution cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic center, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S*angiogenin structures capture how the tRNA substrate is directed by the ribosome next to angiogenins active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, whose abundance increases during cellular stresses.

biochemistry↗

bioRxiv2021

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

During translation, a conserved GTPase elongation factor--EF-G in bacteria or eEF2 in eukaryotes--translocates tRNA and mRNA through the ribosome. 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 various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome*EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by [~]20 [A]. An additional 4-[A] translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. 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.

molecular biology↗

bioRxiv2020

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. Where and how in the elongation cycle +1-frameshifting occurs remains poorly understood. We captured six [~]3.5-[A]-resolution cryo-EM structures of ribosomal elongation complexes formed with the GTPase elongation factor G (EF-G). Three structures with a +1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G, the tRNA shifts to the +1-frame codon near the P site, whereas the freed mRNA base bulges between the P and E sites and stacks on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during mRNA translocation.

molecular biology↗

bioRxiv2020

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of [≥] 20 repeats inhibit the ribosomes peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

biochemistry↗

bioRxiv2019

Extensive ribosome and RF2 rearrangements during translation termination

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S*RF2 structures at up to 3.3 [A] in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long {beta}-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

cell biology↗