Biology subjects

Gentry, R. C.

Publications and source records attributed to Gentry, R. C..

5 recordsLinked to original sources

bioRxiv2026

Extending conventional TIRF microscopy to image single molecules in micromolar analyte backgrounds

Weak, reversible interactions underpin biomolecular recognition, and single-molecule fluorescence (smF) imaging techniques can provide unprecedented insight into those biological processes. Unfortunately, such studies often require micromolar concentrations of fluorophore-labeled biomolecules, which is beyond the accessible range of conventional smF microscopies. Here, we describe a surface-functionalization method based on cloud-point polyethylene glycol (PEG) grafting that enables widefield smF microscopy measurements at micromolar concentrations without the use of nanophotonic devices. Using conventional total internal reflection fluorescence (TIRF) microscopy, we detected single-molecule fluorescence resonance energy transfer (smFRET) from surface-tethered, donor-labeled target molecules with up to 8 micromolar concentrations of freely diffusing, acceptor-labeled analyte molecules in the background--two orders of magnitude higher than typical studies in the literature. Weak, DNA-hybridization and protein-RNA binding equilibria were measured across micromolar range titrations. Together with advances in high-background data analysis, the robust method presented here enables kinetic and thermodynamic analyses of weak biomolecular interactions, especially those limited by nonspecific adsorption and high fluorescence backgrounds, using only standard smF instrumentation.

biophysics↗

bioRxiv2026

Dynamic redistribution of eIF4F controls cap-dependent translation initiation

Translation initiation requires messenger RNAs (mRNAs) to be recognized and loaded into ribosomes through a process catalyzed by the heterotrimeric eukaryotic initiation factor eIF4F. During this process, eIF4F engages the 7-methylguanosine cap at the 5 end of the mRNA and promotes productive engagement with the ribosomal pre-initiation complex (PIC) to facilitate PIC loading onto the mRNA. Although eIF4F is central to translation initiation and its regulation, the molecular mechanism by which eIF4F stimulates PIC loading, and the mechanistic role of the essential ATP hydrolysis step catalyzed by eIF4F, have remained unresolved. Here, we use single-molecule fluorescence microscopy to directly visualize the dynamics of eIF4F during cap recognition and PIC engagement. We show that ATP binding, but not ATP hydrolysis, promotes productive assembly of eIF4F on mRNA and enables dynamic redistribution of eIF4F along the transcript. In contrast, ATP hydrolysis is specifically required for recycling of cap-stalled eIF4F during productive PIC engagement. Furthermore, we identify eIF3 and eIF4B as the minimal PIC-associated factors required to stimulate ATP-hydrolysis-dependent recycling of eIF4F during PIC loading. Together, our results support a model in which productive PIC engagement stimulates ATP-hydrolysis-dependent recycling of eIF4F, thereby coupling eIF4F recycling to PIC loading during translation initiation. This mechanism provides a framework for understanding how mRNA topology, RNA-binding proteins, and the availability of initiation factors can control translational efficiency.

biophysics↗

bioRxiv2024

A dynamic compositional equilibrium governs mRNA recognition by eIF3

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) to drive a diverse set of mechanistic steps during translation of an mRNA into the protein it encodes. And yet, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is in dynamic exchange between the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this dynamic compositional equilibrium and show that mRNA binding by eIF3 is not driven by the full complex but instead by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in mRNA recruitment and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

biophysics↗

bioRxiv2023

The mechanism of mRNA activation

During translation initiation, messenger RNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5 end of the messenger RNA and subsequently activate the message. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and messenger RNA activation remain mysterious. Here, we generate a unique, single-molecule fluorescence imaging system to interrogate the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native messenger RNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the messenger RNA and identify allosteric communication between the eIF4F subunits which ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover novel functions for each subunit of eIF4F and we conclude by presenting a model for messenger RNA activation which precisely defines the composition of the activated message. This model provides a general framework for understanding how messenger RNA molecules may be discriminated from one another, and how other RNA-binding proteins may control the efficiency of translation initiation.

biophysics↗

bioRxiv2023

Cryo-EM studies of the four E. coli paralogs establish ABCF proteins as master plumbers of the peptidyl-transferase center of the ribosome

The genomes of most mesophilic organisms encode multiple ATP-Binding Cassette F (ABCF) proteins. EttA, one of four E. coli paralogs, regulates synthesis of the first peptide bond on the ribosome dependent on ATP/ADP ratio, while Antibiotic Resistance factors (AREs), paralogs in other organisms, both regulate and directly mediate resistance to ribosome-targeted antibiotics. However, the physiological functions remain unclear for most paralogs, and the mechanism-of-action has yet to be rigorously established for any paralog. We herein present single particle cryogenic electron microscopy structures of ribosome complexes of all four E. coli ABCF paralogs (EttA, Uup, YbiT, and YheS), which, together with previously determined ARE structures, show that ABCFs control the binding geometry of the tRNA in the peptidyl-tRNA-binding (P) site on the ribosome. They modulate the position of its acceptor stem relative to the peptidyl transferase center (PTC) in a manner that can either promote (EttA and Uup) or disrupt (YbiT, YheS, and the AREs) proper catalytic geometry. The YbiT/70S reconstructions include a conformation with no density for ribosomal protein bL33, and structural analyses support the exchange of this sub-stoichiometric ribosomal protein being functionally related to conformational changes in YbiT controlled by sequence variations in the strongly non-canonical Signature Sequence in its first ABC domain. Our studies establish general structural/enzymological principles by which the ATPase activity of ABCF proteins controls translation elongation coupled to modulation of conformation and stereochemistry in the catalytic core of the ribosome.

biophysics↗