Biology subjects

Record, T.

Publications and source records attributed to Record, T..

5 recordsLinked to original sources

bioRxiv2025

TDP-43 controls RNA structure through high affinity lattice interactions

TDP-43 is an RNA binding protein implicated in neurodegenerative disease. TDP-43 binds to GU dinucleotide repeats, which are highly abundant sequences in human RNA. Here we show TDP- 43 has one of the highest affinities and specificities measured for an RNA binding protein. Binding prevents formation of the pUG fold, an intramolecular quadruplex, and conversely pUG fold formation prevents TDP-43 binding. A rapid on-rate allows TDP-43 to capture single stranded RNA prior to folding. The protein recognizes the RNA as a 1D lattice, in which overlapping binding sites produce efficient initial binding events that interfere with subsequent interactions. This effect is partially overcome by RNA facilitated protein-protein interactions, which serve to increase the on-rate of a second TDP-43 molecule. In conjunction with all atom models, these data reveal how TDP-43 recognizes RNA repeat sequences and identify an interplay between RNA folding and protein recognition that may be relevant to human disease.

biochemistry↗

bioRxiv2023

Quantifying Amide-Aromatic Interactions at Molecular and Atomic Levels: Experimentally-determined Enthalpic and Entropic Contributions to Interactions of Amide sp2O, N, C and sp3C Unified Atoms with Naphthalene sp2C Atoms in Water

In addition to amide hydrogen bonds and the hydrophobic effect, interactions involving {pi}-bonded sp2 atoms of amides, aromatics and other groups occur in protein self-assembly processes including folding, oligomerization and condensate formation. These interactions also occur in aqueous solutions of amide and aromatic compounds, where they can be quantified. Previous analysis of thermodynamic coefficients quantifying net-favorable interactions of amide compounds with other amides and aromatics revealed that interactions of amide sp2O with amide sp2N unified atoms (presumably C=O{middle dot}{middle dot}{middle dot}H-N hydrogen bonds) and amide/aromatic sp2C (lone pair-{pi}, n-{pi}*) are particularly favorable. Sp3C-sp3C (hydrophobic), sp3C-sp2C (hydrophobic, CH-{pi}), sp2C-sp2C (hydrophobic, {pi}-{pi}) and sp3C-sp2N interactions are favorable, sp2C-sp2N interactions are neutral, while sp2O-sp2O and sp2N-sp2N self-interactions and sp2O-sp3C interactions are unfavorable. Here, from determinations of favorable effects of fourteen amides on naphthalene solubility at 10, 25 and 45 {degrees}C, we dissect amide-aromatic interaction free energies into enthalpic and entropic contributions and find these vary systematically with amide composition. Analysis of these results yields enthalpic and entropic contributions to intrinsic strengths of interactions of amide sp2O, sp2N, sp2C and sp3C unified atoms with aromatic sp2C atoms. For each interaction, enthalpic and entropic contributions have the same sign and are much larger in magnitude than the interaction free energy itself. The amide sp2O-aromatic sp2C interaction is enthalpy-driven and entropically unfavorable, consistent with direct chemical interaction (e.g. lone pair-{pi}) while amide sp3C- and sp2C-aromatic sp2C interactions are entropy-driven and enthalpically unfavorable, consistent with hydrophobic effects. These findings are relevant for interactions involving {pi}-bonded sp2 atoms in protein processes. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/548600v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1683bb4org.highwire.dtl.DTLVardef@27db75org.highwire.dtl.DTLVardef@b915ccorg.highwire.dtl.DTLVardef@1461b04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

E. coli RNA polymerase (2{beta}{beta}{omega}{sigma}70) forms stable open complexes (OC) at {lambda}PR and T7A1 promoters at 37 {degrees}C at similar rates but with very different lifetimes. To probe the origins of these differences, we determine OC lifetimes for full-length (FL) and downstream-truncated (DT) {lambda}PR and T7A1 promoter variants with eight combinations of discriminators and upstream elements. We find the discriminator is the major determinant of OC lifetime, while upstream elements modulate the discriminator effect. The very different lifetimes of these stable OC arise primarily from differences in [Formula], the free energy change for converting I2 (the initial open intermediate) to stable OC. [Formula] is twice as favorable for {lambda}PR-discriminator variants as for T7A1-discriminator variants. Truncation at +6 (DT+6) eliminates most differences in [Formula], destabilizing {lambda}PR-discriminator variants without affecting T7A1-discriminator variants. All DT+6 OC are much more stable and longer-lived than I2. Urea greatly destabilizes OC, primarily affecting RNAP interactions with +6 and upstream DNA. From these findings we deduce that stabilization of I2 involves closing the {beta}-clamp with extensive coupled folding to contact the region of promoter DNA from the transcription start-site to +6. Lifetime differences between DT+6, DT+12 and FL promoters allow dissection of contributions to OC stability from interactions of regions of the downstream duplex with downstream mobile elements (DME; {beta}-lobe, {beta}-clamp, {beta}-jaw). We propose an allosteric network by which differences in discriminator and -10 sequence are sensed by {sigma}1.2 and {beta}-gate loop and transmitted to {sigma}1.1 and {beta}-lobe to affect DME-duplex interactions that determine OC lifetime.

biophysics↗

bioRxiv2020

Kinetic-Mechanistic Evidence for Which E. coli RNA Polymerase-{lambda}PR Open Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

In transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at {lambda}PR promoter at 19{degrees}C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, -10 interactions are disrupted in extending 6-mer to 9-mer, and -35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25{degrees}C and 37{degrees}C. Initiation at 37{degrees}C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37{degrees}C is slower than at 25{degrees}C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37{degrees}C mechanism. Analysis reveals that the 37{degrees}C {lambda}PR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary {lambda}PR OC species below 25{degrees}C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and -10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts. SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with {lambda}PR promoter at 37{degrees}C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and -10 region and with -35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and -10 regions with RNAP.

biochemistry↗

bioRxiv2020

Experimentally-Determined Strengths of Atom-Atom (C, N, O) Interactions Responsible for Protein Self-Assembly in Water: Applications to Folding and Other Protein Processes

Folding and other protein self-assembly processes are driven by favorable interactions between O, N, and C unified atoms of the polypeptide backbone and sidechains. These processes are perturbed by solutes that interact with these atoms differently than water does. C=O{middle dot}{middle dot}{middle dot}HN hydrogen bonding and various {pi}-system interactions have been better-characterized structurally or by simulations than experimentally in water, and unfavorable interactions are relatively uncharacterized. To address this situation, we previously quantified interactions of alkylureas with amide and aromatic compounds, relative to interactions with water. Analysis yielded strengths of interaction of each alkylurea with unit areas of different hybridization states of unified O, N, C atoms of amide and aromatic compounds. Here, by osmometry, we quantify interactions of ten pairs of amides selected to complete this dataset. A novel analysis yields intrinsic strengths of six favorable and four unfavorable atom-atom interactions, expressed per unit area of each atom and relative to interactions with water. The most favorable interactions are sp2O - sp2C (lone pair-{pi}, presumably n-{pi}*), sp2C - sp2C ({pi}-{pi} and/or hydrophobic), sp2O-sp2N (hydrogen bonding) and sp3C-sp2C (CH-{pi} and/or hydrophobic). Interactions of sp3C with itself (hydrophobic) and with sp2N are modestly favorable, while sp2N interactions with sp2N and with amide/aromatic sp2C are modestly unfavorable. Amide sp2O-sp2O interactions and sp2O-sp3C interactions are more unfavorable, indicating the preference of amide sp2O to interact with water. These intrinsic interaction strengths are used to predict interactions of amides with proteins and chemical effects of amides (including urea, N-ethylpyrrolidone (NEP), and polyvinyl-pyrrolidone (PVP)) on protein stability. SignificanceQuantitative information about strengths of amide nitrogen-amide oxygen hydrogen bonds and {pi}-system and hydrophobic interactions involving amide-context sp2 and/or sp3 carbons is needed to assess their contributions to specificity and stability of protein folds and assemblies in water, as well as to predict or interpret how urea and other amides interact with proteins and affect protein processes. Here we obtain this information from thermodynamic measurements of interactions between small amide molecules in water and a novel analysis that determines intrinsic strengths of atom-atom interactions, relative to water and per unit area of each atom-type present in amide compounds. These findings allow prediction or interpretation of effects of any amide on protein processes from structure, and may be useful to analyze protein interfaces.

biochemistry↗