scholarly journals Transcription inhibition by the depsipeptide antibiotic salinamide A

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
David Degen ◽  
Yu Feng ◽  
Yu Zhang ◽  
Katherine Y Ebright ◽  
Yon W Ebright ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Antibacterial Drug ◽  
Transcription Inhibition ◽  
Bacterial Rna ◽  
Cellular Target ◽  
Nucleotide Addition ◽  
And Function

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.

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 The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI)

2020 ◽  
Vol 295 (22) ◽  
pp. 7635-7652
Author(s):  
Christopher D. Radka ◽  
Matthew W. Frank ◽  
Jiangwei Yao ◽  
Jayaraman Seetharaman ◽  
Darcie J. Miller ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Binding Pocket ◽  
Structural Features ◽  
Antibacterial Drug ◽  
Carrier Protein

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix–helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

scholarly journals Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

2016 ◽  
Vol 113 (15) ◽  
pp. 4051-4056 ◽  
Author(s):  
Bin Liu ◽  
Yuhong Zuo ◽  
Thomas A. Steitz
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Active Site ◽  
Transcription Initiation ◽  
De Novo ◽  
Addition Reaction ◽  
Specific Interactions ◽  
Promoter Recognition ◽  
Nucleotide Addition ◽  
Promoter Dna

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

scholarly journals Structural characterization of LsrK to target quorum sensing and comparison between X-ray and homology model

2020 ◽  
Author(s):  
Prasanthi Medarametla ◽  
Tuomo Laitinen ◽  
Antti Poso
Keyword(s):  
Crystal Structure ◽  
Quorum Sensing ◽  
Conformational Changes ◽  
Protein Function ◽  
Bacterial Resistance ◽  
Antibacterial Drug ◽  
Structure Based Drug Design ◽  
Binding Characteristics ◽  
Autoinducer 2 ◽  
Homology Models

ABSTRACTQuorum sensing is being investigated as an alternative therapeutic strategy in antibacterial drug discovery to combat bacterial resistance. LsrK is an autoinducer-2 kinase, playing a key role in the phosphorylation of autoinducer-2 (AI-2) signalling molecules involved in quorum sensing. Inhibiting LsrK could result in reduced pathogenicity by interfering with the quorum sensing signalling. Previously, we have generated homology models to identify LsrK inhibitors using structure-based virtual screening and successfully found the first class of LsrK inhibitors. While conducting these studies, the crystal structure of LsrK was released providing us an opportunity to inspect the reliability and quality of our models. Structural analysis of crystal structure and homology models revealed the consistencies of constructed models with crystal structure in the structural fold and binding site. Further, binding characteristics and conformational changes are investigated using molecular dynamics. These simulations provided us insights into the protein function and flexibility that need to be considered during the structure-based drug design studies targeting LsrK.

scholarly journals Structural basis for transcription initiation by bacterial ECF σ factors

2019 ◽  
Author(s):  
Lingting Li ◽  
Chengli Fang ◽  
Ningning Zhuang ◽  
Tiantian Wang ◽  
Yu Zhang
Keyword(s):  
Crystal Structure ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Specific Gene ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Transcription Initiation Complex ◽  
Promoter Dna ◽  
Context Specific

AbstractBacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

scholarly journals Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

2017 ◽  
Vol 114 (44) ◽  
pp. E9233-E9242 ◽  
Author(s):  
Ananya Ray-Soni ◽  
Rachel A. Mooney ◽  
Robert Landick
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Time Window ◽  
Kinetic Assay ◽  
Termination Rate ◽  
Trigger Loop ◽  
Bacterial Rna ◽  
Nucleotide Addition ◽  
Addition Rate

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA−DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991–1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

scholarly journals Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

2012 ◽  
Vol 287 (15) ◽  
pp. 12353-12364 ◽  
Author(s):  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Yasser A. Mahmmoud ◽  
Elisha J. Hamilton ◽  
Keyvan Karimi Galougahi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Membrane Potential ◽  
Angiotensin Ii ◽  
Conformational Changes ◽  
Ventricular Myocytes ◽  
Conformational State ◽  
Side Chain ◽  
Voltage Dependent ◽  
Cytosolic Glutathione ◽  
And Function

Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump.

scholarly journals Structural studies of Mot1 and NC2 in transcription initiation process

2014 ◽  
Vol 70 (a1) ◽  
pp. C1396-C1396
Author(s):  
Agata Butryn ◽  
Jan Schuller ◽  
Gabriele Stöhr ◽  
Friedrich Förster ◽  
Karl-Peter Hopfner
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Promoter Regions ◽  
Protein Nucleic Acid ◽  
Promoter Dna ◽  
Tata Box Binding Protein

Regulation of protein–nucleic acid interactions plays a key role in various cellular processes. The SNF2/SWI2 protein family forms a large and diverse class of proteins and multiprotein assemblies, which use energy derived from ATP hydrolysis to disrupt or modify protein-DNA interactions. They possess a conserved helicase-like domain accompanied by protein-specific targeting and regulation regions. The SNF2/SWI2 family member Mot1 (Modifier of transcription 1, also known as BTAF1) is a single polypeptide ATP-dependent remodeler. Mot1 acts directly on TBP (TATA-box binding protein) regulating RNA polymerase II preinitiation complex formation in the first stages. Global distribution of TBP on promoter regions is also modulated by NC2 (Negative Cofactor 2). It has been suggested that Mot1 and NC2 can co occupy the same promoter sites influencing the assembly of the transcription machinery simultaneously. Our understanding of the molecular mechanism of SWI2/SNF2 family ATPases is very limited. Previously, we have reported the crystal structure of the complex of Encephalitozoon cuniculi N terminal domain of Mot1 (Mot1NTD) with its substrate, TBP [1]. Here we present the crystal structure of the TBP NC2 Mot1NTD complex bound to a promoter DNA fragment at 3.9Å resolution. In our studies we have applied a combined structural biology approach using crystallography, electron microscopy reconstructions and chemical cross-linking. We probed the conformational changes of the complex during the ATP hydrolysis cycle and unveiled the structural basis of the Mot1–NC2 interplay. Our findings greatly contribute not only to our limited understanding of Mot1 action, but all SNF2/SWI2 family remodeling enzymes.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

Computer-aided structural and molecular insights into the mechanisms by which Pseudouridimycin (PUM) disrupts cleft extension in bacterial RNA polymerase to block DNA entry and exit

2020 ◽  
Vol 17 ◽  
Author(s):  
Ali H. Rabbad ◽  
Fisayo A. Olotu ◽  
Mahmoud E. Soliman
Keyword(s):  
Rna Polymerase ◽  
Bacterial Infections ◽  
Entry And Exit ◽  
Dynamic Binding ◽  
Binding Behavior ◽  
Bacterial Rna ◽  
Nucleotide Addition ◽  
Switch Regions ◽  
Cleft Extension ◽  
Key Residues

Background: The ability of Pseudouridimycin (PUM) to occupy the nucleotide addition site of bacterial RNA Polymerase (RNAP) underlies its inhibitory potency as previously reported. PUM has gained high research interest as a broad-spectrum nucleoside analog that has demonstrated exciting potentials in treating drug-resistant bacterial infections. Objective: Herein, we identified, for the first time, a novel complementary mechanism by which PUM elicits its inhibitory effects on bacterial RNAP. Methods: The dynamic binding behavior of PUM to bacterial RNAP was studied using various dynamic analyses approaches. Results and Discussion: Findings revealed that in addition to occupying the nucleotide addition site, PUM also interrupts the unimpeded entry and exit of DNA by reducing the mechanistic extension of the RNAP cleft and perturbing the primary conformations of the switch regions. Moreover, PUM binding reduced the distances between key residues in the β and β’ subunits that extend to accommodate the DNA. Conclusion: This study’s findings present structural insights that would contribute to the structure-based design of potent and selective PUM inhibitors.

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