Biology subjects

Dutagaci, B.

Publications and source records attributed to Dutagaci, B..

6 recordsLinked to original sources

bioRxiv2023

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 A using a free-electron laser

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

biophysics↗

bioRxiv2023

Binding of Small Molecule Inhibitors to RNA Polymerase-Spt5 Complex Impacts RNA and DNA Stability

Spt5 is an elongation factor that associates with RNA polymerase II (Pol II) during transcription and has important functions in promoter-proximal pausing and elongation processivity. Spt5 was also recognized for its roles in the transcription of expanded-repeat genes that are related to neurodegenerative diseases. Recently, a set of Spt5-Pol II small molecule inhibitors (SPIs) were reported, which selectively inhibit mutant huntingtin gene transcription. Inhibition mechanisms as well as interaction sites of these SPIs with Pol II and Spt5 are not entirely known. In this study, we predicted the binding sites of three selected SPIs at the Pol II-Spt5 interface by docking and molecular dynamics simulations. Two molecules out of three demonstrated strong binding with Spt5 and Pol II, while the other molecule was more loosely bound and sampled multiple binding sites. Strongly bound SPIs indirectly affected RNA and DNA dynamics at the exit site as DNA became more flexible while RNA was stabilized by increased interactions with Spt5. Our results suggest that the transcription inhibition mechanism induced by SPIs can be related to Spt5-nucleic acid interactions, which were altered to some extent with strong binding of SPIs.

biophysics↗

bioRxiv2023

Complex Conformational Space of RNA Polymerase II C-Terminal Domain upon Phosphorylation

Intrinsically disordered proteins (IDPs) have been closely studied during the past decade due to their importance for many biological processes. The disordered nature of this group of proteins makes it difficult to observe its full span of the conformational space either using experimental or computational studies. In this article, we explored the conformational space of the C-terminal domain (CTD) of RNA polymerase II (Pol II), which is also an intrinsically disordered low complexity domain, using enhanced sampling methods. We provided a detailed conformational analysis of model systems of CTD with different lengths; first with the last 44 residues of the human CTD sequence and finally the CTD model with two heptapeptide repeating units. We then investigated the effects of phosphorylation on CTD conformations by performing simulations at different phosphorylated states. We obtained broad conformational spaces in non-phosphorylated CTD models and phosphorylation has complex effects on the conformations of the CTD. These complex effects depend on the length of the CTD, spacing between the multiple phosphorylation sites, ion coordination and interactions with the nearby residues.

biophysics↗

bioRxiv2022

Characterization of RNA Polymerase II Trigger Loop Mutations using Molecular Dynamics Simulations and Machine Learning

Catalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that lethal mutations tended to increase distances of TL residues to the NTP substrate, while viable loss-of-function (LOF) substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, GOF mutants generally have a disrupting effect on hydrophobic contacts among TL and nearby helices. AUTHOR SUMMARYRNA polymerase II (Pol II) synthesizes RNA with the help of an active site domain called trigger loop (TL). The mutations of TL cause changes in the activity of Pol II that could range from gain-of function (GOF) to loss-of-function (LOF) or lethal. This study provides a systematic characterization of the structural and functional outcomes of the TL mutations using molecular dynamics (MD) simulations and machine learning (ML). We obtained functional phenotypes of mutants by ML using the genetic fitness scores as the input. We revealed that mutant TL sequences could predict the functional outcomes at a relatively high correlation. Then, we performed MD simulations to relate the structural information to the phenotypes. The analysis of the MD data suggested that the lethal mutants had increased distances between the TL and the substrate, while a subset of LOF mutants showed increased distances between TL and another active site domain called bridge helix (BH). On the other hand, GOF mutants had effects on the hydrophobic interactions around the active site. Overall, this study enhances our understanding of the effects of TL mutations to the Pol II function.

biophysics↗

bioRxiv2022

Protein-Nucleic Acid Interactions for RNA Polymerase II Elongation Factors by Molecular Dynamics Simulations

RNA polymerase II (Pol II) forms a complex with elongation factors to proceed the elongation stage of the transcription process. In this work, we studied elongation factor SPT5 and explored protein nucleic acid interactions for the isolated systems of KOW1 and KOW4 domains of SPT5 with DNA and RNA, respectively. We performed molecular dynamics (MD) simulations using three commonly used force fields that are CHARMM c36m, AMBER ff14sb and ff19sb. These simulations showed that most of the protein-nucleic acid interactions in the native state were retained with an increased electrostatic binding free energy for all force fields used. RNA was found highly dynamic with all force fields while DNA had relatively more stable conformations with the AMBER force fields compared to CHARMM. Furthermore, we performed MD simulations of the complete elongation complex using CHARMM c36m force field to compare the dynamics and interactions in the isolated systems. Similar strong KOW1 and DNA interactions were observed in the complete elongation complex simulations and DNA was further stabilized by a network of interactions involving SPT5-KOW1, SPT4 and rpb2 of Pol II. Overall, our study showed that the accuracy of force fields and the presence of the entire interaction network are important for elucidating the dynamics of protein-nucleic acid systems.

biophysics↗

bioRxiv2020

Charge-Driven Phase Separation of RNA and Proteins without Disorder

Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well established drivers of liquid-liquid phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Condensates retain liquid character and phase diagrams are mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components. STATEMENT OF SIGNIFICANCELiquid-liquid phase separation has been recognized as a key mechanism for forming membrane-less organelles in cells. Commonly discussed mechanisms invoke a role of disordered peptides and specific multi-valent interactions. We report here phase separation of RNA and proteins based on a more universal principle of charge complementarity that does not require disorder or specific interactions. The findings are supported by coarse-grained simulations, theory, and experimental validation via microscopy and spectroscopy. The broad implication of this work is that condensate formation may be a universal phenomenon in biological systems.

biophysics↗