Biology subjects

Sosa, R. P.

Publications and source records attributed to Sosa, R. P..

4 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 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↗

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↗

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↗