Biology subjects

Bustamante, C. J.

Publications and source records attributed to Bustamante, C. J..

9 recordsLinked to original sources

bioRxiv2026

FoxO1 and HNF1α cooperatively remodel nucleosome architecture and mechanical stability to activate transcription

How different transcription factors (TFs) cooperate to bind nucleosomal DNA targets remains unclear. Cryo-EM and force spectroscopy show that the pioneer factor FoxO1 binds superhelical location (SHL) +2 of nucleosomes assembled on the human IGFBP1 promoter, enhances distal DNA unwrapping, destabilizes the histone core, and reduces nucleosome mechanical stability. TF HNF1 binds SHL +5/+7 as a dimer, induces extensive DNA unwrapping without perturbing the histone core, and reshapes nucleosome disassembly dynamics. FoxO1 enhances HNF1 recruitment, and together they stabilize the nucleosome during its mechanically-induced disassembly trajectories. In-cell nucleosome footprinting on active promoters shows that FoxO1 binding enhances unwrapped nucleosomes and subnucleosomes presence, while HNF1 increases stabilized and breathing nucleosomes and reduces the subnucleosome population. Combined, they display an intermediate nucleosome phenotype. Our data leads to a model where FoxO1 destabilizes the nucleosome and enhances HNF1 recruitment, and together they remodel nucleosome architecture both in vitro and in native chromatin.

biophysics↗

bioRxiv2026

The Rossmann2x2 Fold Attains its Native Structure Via a Defined Pathway of Sequential and Cooperative Folding Units

Despite progress in predicting protein structures, how proteins arrive at their native state remains a subject of continuous debate. We present a single molecule force spectroscopy study of conserved Rossmann2x2 fold. Using novel single-molecule methods, we follow in real time and annotate the unfolding and refolding intermediates of Ross, a representative of this topology. This protein folds along a single reversible pathway involving the ordered and sequential organization of discrete and cooperative folding units or foldons. This strict order of events results from the formation of an initial, autonomously folding unit (primary foldon) followed by the subsequent organization of secondary foldons whose stabilities depend on their interactions with previously organized ones. This process, if generalized, represents a great simplification of the protein folding mechanism, as identification of foldons in nature and their folding hierarchy, should allow prediction of the folding pathway of any natural protein from its sequence.

biophysics↗

bioRxiv2026

The Central Coupler of the AAA+ ATPase ClpXP Controls Intersubunit Communication and Couples the Conversion of Chemical Energy into the Generation of Force

ClpX is a clockwise hexameric helical arrangement that hydrolyzes ATP to unfold proteins and translocate them into the proteolytic chamber. We investigate the central coupler, a three -helix module conserved among AAA+ ATPases, which is proposed to enable intersubunit communication and mechanochemical coupling. Although fundamental in AAA+ ATPases, the molecular mechanism underlying these processes remains elusive in ClpX. By combining single-molecule optical tweezers, biochemical assays and single particle cryo-EM we demonstrate that the central coupler, of the second-highest subunit of the ClpX clockwise helical arrangement, positions its Arginine-finger, sensor-I and sensor-II to contact the ATP of the anticlockwise neighbor subunit to trigger its hydrolysis. Then, the central coupler of the highest subunit rapidly couples the ATP hydrolysis with the downward motion of its substrate-translocating loop, enabling fast force generation to swiftly unfold protein substrates while maximizing the thermodynamic efficiency of the motor.

biophysics↗

bioRxiv2026

Spt5's central KOW domains and the Pol II Stalk Collaborate to Regulate Chromatin and 3'-End Processing

Spt5 is a universally conserved multidomain transcription elongation factor that acts as a component of all Pol II elongation complexes. Structural studies indicate that several of Spt5s central KOW domains lie adjacent to the Pol II stalk, composed of subunits Rpb4 and Rpb7. However, their in vivo functions are unknown. Here we show that Spt5 and Rpb4/7 jointly modulate 3-end formation and co-transcriptional chromatin integrity in Saccharomyces cerevisiae. We identify mutations in the SPT5 KOW2-3 domains and RPB7 that cause cryptic initiation of transcription and alter 3-end formation of RNA transcripts. Molecular readthrough assays reveal allele-specific changes at both GAL10 and SNR13, consistent with impacts on CPF/CF- and NNS-dependent termination. Proteomic experiments with isolated KOW2-3 domain enrich factors from both pathways as well as chromatin regulators, overlapping known Rpb7 interactors. Together, these findings support a model in which Spt5 KOW2-3/Pol II stalk region acts as a recruitment platform that coordinates pre-mRNA processing and chromatin dynamics during elongation, revealing new roles for the central KOW domains of Spt5. SummaryThis work describes a cooperative in vivo function for Spt5s central KOW domains and the Pol II stalk in Saccharomyces cerevisiae. Allele-specific genetics and reporter assays show cooperative effects of SPT5 and RPB4/7 on cryptic initiation and 3'-end formation; double-mutant analyses reveal synthetic interactions. RT-qPCR at GAL10 and SNR13 demonstrates regulation of both poly(A) and non-coding transcript termination. Spt5 KOW pull-down proteomics enrich poly(A) and non-coding termination factors, as well as chromatin regulators that overlap with known Rpb7 interactors. Together, the data support a model in which Spt5 and the Pol II stalk coordinate chromatin integrity and termination during elongation.

genetics↗

bioRxiv2023

Secondary Structure Detection Through Direct Nanopore RNA Sequencing

Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RTs dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.

biophysics↗

bioRxiv2022

Actin-driven protrusions generate rapid long-range membrane tension propagation in cells

Membrane tension is thought to be a long-range integrator of cell physiology. This role necessitates effective tension transmission across the cell. However, the field remains strongly divided as to whether cell membranes support or resist tension propagation, in part due to a lack of adequate tools for locally manipulating membrane tension. We overcome these limitations by leveraging optogenetics to generate localized actinbased protrusions while concurrently monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions elicit rapid global membrane tension propagation with little to no attenuation, while forces applied to the cell membrane only do not. We present a simple unifying mechanical model in which mechanical forces that act on both the membrane and actin cortex drive rapid, robust membrane tension propagation. SummaryMechanical perturbations acting on both actin cortex and plasma membrane drive global membrane tension increase within seconds

cell biology↗

bioRxiv2022

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetrical occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection we show that the low-force transition corresponds to the undoing of the outer-wrap of one side of the nucleosome (~27 bp), a process that can occur either cooperatively or non-cooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ~76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition, and another that coincides but does not contribute to the high-force transition. Significance statementNucleosomes, the fundamental structural unit of chromatin, consists of ~147 DNA base pairs wrapped around a histone protein octamer. Determining the forces required to unwrap the DNA from the core particle and the stepwise transitions involved in the process are essential to characterize the strength of the nucleosomal barrier and its contribution as a mechanism of control of gene expression. Here, we performed combined optical tweezers and single-molecule fluorescence measurements to annotate the specific DNA segments unwrapping during the force transitions observed in mechanical unwrapping of nucleosomes. Furthermore, we characterize the mechanical signatures of subnucleosomal particles: hexasomes and tetrasomes. The characterization performed in this work is essential for the interpretation of ongoing studies of chromatin remodelers, polymerases, and histone chaperones.

biophysics↗

bioRxiv2022

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ( reverse-topology), but they can also be directed towards the cytosol ( normal-topology). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA+ ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA+ ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.

biophysics↗

bioRxiv2020

Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation

During transcription initiation, the RNA polymerase holoenzyme (RNAP) and the promoter form an open complex. For many promoters, this interaction involves upstream DNA wrapping, downstream promoter bending, and associated large-scale protein rearrangements. Although these processes have been reported across the life kingdom, their structure, energetics, and role in transcription remain an area of active research. Using optical tweezers, we find that these processes become energetically and reversibly coupled after the formation of the open promoter complex, providing the main contribution to their stability. Using electron microscopy and single particle analysis, we find that the interaction encompasses from positions -76 to +18 along the template, that it involves an overall DNA bent angle of ~245{degrees}, and that the upstream wrapping is enabled by interactions between the C-terminal domains of RNAPs alpha subunits and proximal and middle upstream promoter regions. The energy associated with upstream wrapping, downstream bending and its coupling to downstream rearrangements does not require specific upstream promoter sequence, and correlate positively with the rate of transcription DNA bubble formation as reported by a real-time fluorescence assay. Our results suggest that the coupling between upstream and downstream events are part of a cis-regulatory network established after the opening of the DNA bubble, that could furnish a control mechanism of gene expression by protein factors and regulatory metabolites. SummaryThe first step of gene expression involves transcription of DNA into RNA by RNA polymerase (RNAP). RNAP recognizes a promoter sequence forming the transcriptionally active open complex. For several promoters, DNA wraps around the RNAP. We find that upstream wrapping contacts are energetically coupled and occur cooperatively with downstream rearrangements in the open complexes, providing the largest contribution to their stability. We also determined that upstream wrapping is enabled by interactions between non-specific upstream promoter regions and RNAP subunit C-terminal domains. Significantly, the strength of these contacts correlates with the rate of DNA bubble opening, and is regulated by factors such as the transcriptional regulator ppGpp. We suggest that any modulator altering upstream wrapping and downstream rearrangements could finely tune gene expression in response to the needs of the cell

biophysics↗