Biology subjects

Harris, G. M.

Publications and source records attributed to Harris, G. M..

4 recordsLinked to original sources

bioRxiv2025

Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10

The evolutionarily conserved methyltransferase Trm10 catalyzes N1 methylation of guanosine 9 (G9) in select tRNAs, but the basis of specific substrate recognition and modification has remained unclear. Using an S-adenosyl-L-methionine analog, we trapped a post-catalytic state of the Trm10-tRNAGly complex for structure determination by cryogenic electron microscopy. Three distinct complexes were captured: two monomeric Trm10-tRNA complexes with distinct tRNA acceptor stem orientations ("closed" and "open"), and a minor dimeric complex with two Trm10s bound to the same tRNA. The monomeric structures identify conserved residues involved in tRNA interactions across a positively charged surface that guide G9 into the catalytic site and stabilize the flipped nucleotide. In the tRNAopen conformation, acceptor stem rotation weakens tRNA-protein contacts, consistent with a product-release state. The dimeric complex, supported by tRNA-dependent protein crosslinking and molecular dynamics (MD) simulations, positions one Trm10 on G9 similarly to the monomeric complexes, while the other contacts distal tRNA regions, suggesting a functional role in promoting functionally critical conformational transition(s). MD simulations also show how Trm10 achieves selective stabilization of G9 over A9 in the binding pocket. Overall, our findings reveal the mechanism of G9-specific tRNA methylation by Trm10 and suggest a unique mechanism of action among RNA-modifying SPOUT methyltransferases.

biochemistry↗

bioRxiv2025

Mechanism of 30S subunit recognition and modification by the conserved bacterial ribosomal RNA methyltransferase RsmI

Ribosomal RNA (rRNA) modifications are important for ribosome function and can influence bacterial susceptibility to ribosome-targeting antibiotics. The universally conserved 16S rRNA nucleotide C1402, for example, is the only 2-O-methylated nucleotide in the bacterial small (30S) ribosomal subunit and this modification fine tunes the shape and structure of the peptidyl tRNA binding site. The Cm1402 modification is incorporated by the conserved bacterial 16S rRNA methyltransferase RsmI, but it is unclear how RsmI is able to recognize its 30S substrate and specifically modify its buried target nucleotide. We determined a 2.42 [A] resolution cryo-EM structure of the RsmI-30S complex and, with accompanying functional analyses, show that RsmI anchors itself to the 30S subunit through multiple contacts with a conserved 16S rRNA surface previously only seen in the assembled subunit. This positions RsmI to induce an extensive h44 distortion to access C1402 that is unprecedented among 16S rRNA methyltransferases characterized to date. These analyses also reveal an essential contribution to 30S subunit interaction made by the previously structurally uncharacterized RsmI C-terminal domain, RsmI-induced RNA-RNA interactions with C1402, and an unappreciated dependence on a divalent metal ion for activity that suggests RsmI may be first of a distinct class of metal- and SAM-dependent RNA O-methyltransferases. This study significantly expands our mechanistic understanding of how intrinsic bacterial methyltransferases like RsmI modify their rRNA targets. Further, recognition of distant ribosome features and extensive unfolding of a critical rRNA functional center point to a potential role in accurate 30S subunit biogenesis.

biochemistry↗

bioRxiv2024

Enhanced Piezoelectric Performance of PVDF-TrFE Nanofibers through Annealing for Tissue Engineering Applications

This study investigates bioelectric stimulations role in tissue regeneration by enhancing the piezoelectric properties of tissue-engineered grafts using annealed poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) scaffolds. Annealing at temperatures of 80{degrees}C, 100{degrees}C, 120{degrees}C, and 140{degrees}C was assessed for its impact on material properties and physiological utility. Analytical techniques such as Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction (XRD) revealed increased crystallinity with higher annealing temperatures, peaking in {beta}-phase content and crystallinity at 140{degrees}C. Scanning Electron Microscopy (SEM) showed that 140{degrees}C annealed scaffolds had enhanced lamellar structures, increased porosity, and maximum piezoelectric response. Mechanical tests indicated that 140{degrees}C annealing improved elastic modulus, tensile strength, and substrate stiffness, aligning these properties with physiological soft tissues. In vitro assessments in Schwann cells demonstrated favorable responses, with increased cell proliferation, contraction, and extracellular matrix attachment. Additionally, genes linked to extracellular matrix production, vascularization, and calcium signaling were upregulated. The foreign body response in C57BL/6 mice, evaluated through Hematoxylin and Eosin (H&E) and Picrosirius Red staining, showed no differences between scaffold groups, supporting the potential for future functional evaluation of the annealed group in tissue repair.

bioengineering↗

bioRxiv2019

Promoting neuronal outgrowth using ridged scaffolds coated with extracellular matrix proteins.

Spinal cord injury (SCI) results in cell death, demyelination, and axonal loss. The spinal cord has a limited ability to regenerate and current clinical therapies for SCI are not effective in helping promote neurologic recovery. We have developed a novel scaffold biomaterial that is fabricated from the biodegradable hydrogel oligo[poly(ethylene glycol)fumarate] (OPF). We have previously shown that positively charged OPF scaffolds (OPF+) in an open spaced, multichannel design can be loaded with Schwann cells to support axonal generation and functional recovery following SCI. We have now developed a hybrid OPF+ biomaterial that increases the surface area available for cell attachment and that contains an aligned microarchitecture and extracellular matrix (ECM) proteins to better support axonal regeneration. OPF+ was fabricated as 0.08 mm thick sheets containing 100 m high polymer ridges that self-assembles into a spiral shape when hydrated. Laminin, fibronectin, or collagen I coating promoted neuron attachment and axonal outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and neurites aligned in the direction of the ridge. Schwann cells seeded on laminin coated OPF+ sheets aligned along the ridges over a 6-day period and could myelinate dorsal root ganglion neurons over 4 weeks. The OPF+ sheets support axonal regeneration when implanted into the transected spinal cord. This novel scaffold design, with closer spaced ridges and Schwann cells is a novel biomaterial construct to promote regeneration after SCI.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC=\"FIGDIR/small/788539v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1ea0ae3org.highwire.dtl.DTLVardef@70e1f7org.highwire.dtl.DTLVardef@1034b77org.highwire.dtl.DTLVardef@14f784d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗