scholarly journals TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

2016 ◽  
Vol 113 (13) ◽  
pp. E1816-E1825 ◽  
Author(s):  
Sarah Schulz ◽  
Andreas Gietl ◽  
Katherine Smollett ◽  
Philip Tinnefeld ◽  
Finn Werner ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Conformational Space ◽  
Single Molecule Fret ◽  
Transcription Complexes ◽  
Basal Transcription Factors ◽  
Transcription Cycle

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Small-Molecule Hormones: Molecular Mechanisms of Action

2013 ◽  
Vol 2013 ◽  
pp. 1-21 ◽  
Author(s):  
Monika Puzianowska-Kuznicka ◽  
Eliza Pawlik-Pachucka ◽  
Magdalena Owczarz ◽  
Monika Budzińska ◽  
Jacek Polosak
Keyword(s):  
Transcription Factors ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Hormone Receptors ◽  
Target Genes ◽  
Direct Interaction ◽  
Nuclear Hormone Receptors ◽  
Membrane Phospholipids ◽  
Basal Transcription Factors ◽  
Hormonal Stimulus

Small-molecule hormones play crucial roles in the development and in the maintenance of an adult mammalian organism. On the molecular level, they regulate a plethora of biological pathways. Part of their actions depends on their transcription-regulating properties, exerted by highly specific nuclear receptors which are hormone-dependent transcription factors. Nuclear hormone receptors interact with coactivators, corepressors, basal transcription factors, and other transcription factors in order to modulate the activity of target genes in a manner that is dependent on tissue, age and developmental and pathophysiological states. The biological effect of this mechanism becomes apparent not earlier than 30–60 minutes after hormonal stimulus. In addition, small-molecule hormones modify the function of the cell by a number of nongenomic mechanisms, involving interaction with proteins localized in the plasma membrane, in the cytoplasm, as well as with proteins localized in other cellular membranes and in nonnuclear cellular compartments. The identity of such proteins is still under investigation; however, it seems that extranuclear fractions of nuclear hormone receptors commonly serve this function. A direct interaction of small-molecule hormones with membrane phospholipids and with mRNA is also postulated. In these mechanisms, the reaction to hormonal stimulus appears within seconds or minutes.

scholarly journals Pausing controls branching between productive and non-productive pathways during initial transcription

2017 ◽  
Author(s):  
David Dulin ◽  
David L. V. Bauer ◽  
Anssi M. Malinen ◽  
Jacob J. W. Bakermans ◽  
Martin Kaller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Tightly Coupled ◽  
Relationship Of ◽  
The Relationship

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

scholarly journals Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

2021 ◽  
Author(s):  
Joshua C. Sanders ◽  
Erik D. Holmstrom
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Resonance Energy ◽  
Integrative Approach ◽  
Biological Macromolecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Biomolecular Simulations ◽  
Diverse Interactions

Abstract The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole–dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).

scholarly journals Single Molecule FRET Reveals Lipid Induced Conformational Changes in Cytoplasmic Domain of Kir2.1

2019 ◽  
Vol 116 (3) ◽  
pp. 109a
Author(s):  
Joshua B. Brettmann ◽  
Sun Joo Lee ◽  
Shizhen Wang ◽  
Colin G. Nichols
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Single Molecule Fret

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

scholarly journals Insights into the substrate binding mechanism of SULT1A1 through molecular dynamics with excited normal modes simulations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Balint Dudas ◽  
Daniel Toth ◽  
David Perahia ◽  
Arnaud B. Nicot ◽  
Erika Balog ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Normal Modes ◽  
Md Simulations ◽  
Conformational Space ◽  
Metabolizing Enzymes ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Excited Normal Modes

AbstractSulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein–ligand interactions studies where major rearrangements are expected.

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