Biology subjects

Showalter, S. A.

Publications and source records attributed to Showalter, S. A..

5 recordsLinked to original sources

bioRxiv2025

Residue-level mapping of crowding effects on protein phase separation

Proteins can undergo liquid-liquid phase separation to form condensates within the crowded cellular environment. In vitro, polymer-based crowding agents are widely used to mimic this effect, but how their chemical environments influence phase separation compared to native cytosol remains unclear. Here, we use NMR to probe the chemical influences on the intrinsically disordered region of RNA polymerase II under different crowding conditions, including polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol. We find a general trend of enhanced protein self-interactions across all conditions, but also distinct chemical environments that depend on crowder identity, reflecting changes in preferential interactions. Given the widespread use of polymer crowders in phase separation studies, our results establish a strategy to dissect the microscopic role of crowders and lay the foundation for designing more physiologically relevant in vitro crowding models of protein phase separation. More broadly, this framework enables systematic probing of residue-level environmental influences in complex settings including within the cellular milieu.

biophysics↗

bioRxiv2025

RNA polymerase II CTD Ser5 phosphorylation induces competing effects of expansion and compaction

The carboxy-terminal domain (CTD) of RNA Polymerase II, composed of tandem heptad repeats with the consensus sequence YSPTSPS, orchestrates the transcription cycle through a dynamic series of post-translational modifications. Among these, the phosphorylation of Ser5 is critical for initiator/promoter clearance and the recruitment of capping enzymes. However, the exact conformational consequences of these modifications are still not fully understood. This study investigates how Ser5 phosphorylation affects the local and global conformation of the CTD, its influence on proline isomerization, and how variations in the repeat sequence modulate these effects. We employed Gaussian accelerated Molecular Dynamics (GaMD) simulations on 3-heptad models of both the consensus CTD sequence and an Asn7 variant. We found that Ser5 phosphorylation promotes expansion of the peptide due to the repulsion between the negatively-charged phosphate groups, but also increases the population of cis-Pro6, which leads to compaction. We used a clustering algorithm to identify commonly populated conformations, with a focus on those conformations that change in population with Ser5 phosphorylation. Our simulations reveal that the expansion of the CTD due to Ser5 phosphorylation is accompanied by a change in local, intra-heptad interactions in both variants. Notably, phosphorylation significantly increases the population of cis-Pro6 due to steric repulsion between the Asn7 side chain and the large side chain of the phosSer5, but has a smaller increase in the consensus variant. These results clarify the underlying mechanisms by which phosphorylation can modulate the CTDs structural landscape to regulate the transcription cycle. SignificanceThe RNA Polymerase II CTD is a critical part of the machinery that regulates transcription, and therefore, understanding how it functions in this process is essential. However, the conformational effects of known modifications to the CTD, such phosphorylation and proline isomerization, are not fully understood. This paper uses all-atom molecular dynamics simulations to identify the specific conformational changes to the disordered CTD with phosphorylation, and with changing heptad sequence. We also identify the interactions that are responsible for these changes. Our results emphasize that two chemical properties of phosphate groups, their negative charge and their large size, can affect protein conformation. For the CTD, these properties have competing effects on the overall compaction of the disordered sequence.

biophysics↗

bioRxiv2025

Phosphorylation patterns modulate the transient secondary structure of RNA polymerase II CTD without altering its global conformation

The intrinsically disordered C-terminal domain (CTD) of RNA polymerase II coordinates transcription and co-transcriptional events through dynamic phosphorylation patterns. While it has been long hypothesized that phosphorylation induces structural changes in the CTD, a direct comparison of how different phosphorylation patterns modulate the CTD conformation has been limited. Here, we generated two distinct phosphorylation patterns in an essential Drosophila CTD region with the kinase Dyrk1a: one where Ser2 are primarily phosphorylated, mimicking the state near transcription termination, and a hyperphosphorylation state where most Ser2, Ser5, and Thr4 residues are phosphorylated, expanding on our work on Ser5 phosphorylation, which mimics early transcription elongation. Using 13C Direct-Detect NMR, we show that the CTD has a tendency to form transient beta strands and beta turns, which is altered differently by Ser2 and Ser5 phosphorylation. Small angle x-ray scattering (SAXS) revealed no significant changes in the CTD global dimensions even at high levels of phosphorylation, contradicting the common assumption of phosphorylation-induced chain expansion. Our findings support a transient beta model in which unphosphorylated CTD adopts transient beta strands at Ser2 during transcription pre-initiation. These transient structures are disrupted by Ser5 phosphorylation in early elongation, and later restored by Ser2 phosphorylation near termination for recruiting beta turn-recognizing termination factors.

biophysics↗

bioRxiv2024

Molecular insights into the interaction between a disordered protein and a folded RNA

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation. SIGNIFICANCESubcellular organization through the formation of biomolecular condensates has emerged as an important contributor to myriad cellular functions, with implications in homeostasis, stress response, and disease. To understand the general and specific principles that support condensate formation, we must interrogate the interactions and assembly of their constituent biomolecules. To this end, this study introduces a simple model system comprised of a small, disordered protein and small RNA that undergo charge-driven, associative phase separation. In addition to extensive biophysical characterization of these molecules and their complex, we also generate new insights into mode of interaction and assembly between an unstructured protein and a structured RNA.

biochemistry↗

bioRxiv2020

An adjunctive therapy approach prevents antibiotic resistance emergence in opportunistic pathogens colonizing the gut

A key challenge in antibiotic stewardship is figuring out how to use antibiotics therapeutically without promoting the evolution of antibiotic resistance. Here, we demonstrate proof of concept for an adjunctive therapy that allows intravenous antibiotic treatment without driving the evolution and onward transmission of resistance. We repurposed the FDA-approved bile sequestrant cholestyramine, which we show binds the antibiotic daptomycin, as an anti-antibiotic to disable systemically-administered daptomycin reaching the gut. We hypothesized that adjunctive cholestyramine could enable therapeutic daptomycin treatment in the bloodstream, while preventing transmissible resistance emergence in opportunistic pathogens colonizing the gastrointestinal tract. We tested this idea in a mouse model of Enterococcus faecium gastrointestinal tract colonization. In mice treated with daptomycin, adjunctive cholestyramine therapy reduced the fecal shedding of daptomycin-resistant E. faecium by up to 80-fold. These results provide proof of concept for an approach that could reduce the spread of antibiotic resistance for important hospital pathogens.

evolutionary biology↗