scholarly journals Mechanism of upstream promoter element stimulation of transcription at a ribosomal RNA promoter determined by single-molecule imaging

2020 ◽  
Author(s):  
Jeffrey P. Mumm ◽  
Larry J. Friedman ◽  
Jeff Gelles
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Kinetic Mechanism ◽  
Initiation Rate ◽  
Α Subunit ◽  
Kinetic Mechanisms ◽  
Up Elements ◽  
Initial Binding ◽  
Stimulation Of

AbstractDNA elements upstream of transcription promoters play a role in regulating transcription initiation in all organisms. In bacteria, upstream A-T rich sequences called UP elements can stimulate transcription through contact with the α subunit C-terminal domain (αCTD) of core RNA polymerase (RNAP), but the kinetic mechanisms by which they do so remain unclear. We investigated the role of the UP element in stimulating initiation from the strong E. coli 16s rRNA promoter using single-molecule fluorescence microscopy to visualize σ70RNAP holoenzyme binding and the formation nascent RNA by oligonucleotide probe hybridization on individual DNA molecules containing the rrnB P1 promoter. By directly detecting initial binding of σ70RNAP to promoter and monitoring the lifetimes of promoter-polymerase complexes, the experiments reveal the kinetic mechanism of polymerase recruitment to the promoter and the subsequent conformational change that stabilizes binding. The presence of UP stimulated the rate of initial binding of polymerase to promoter by at least six-fold, and this stimulation was fully sufficient to account for the increase in initiation rate by UP. Thus, UP likely functions at this strong promoter simply by acting as a binding target for the rapidly reorienting αCTD domain tethered to the core polymerase. In contrast, there were only minor effects of UP on the measured rates of the conformational change or the dissociation rates of the initial σ70RNAP promoter complexes. These studies define a paradigmatic kinetic mechanism for stimulation of transcription initiation by direct αCTD-DNA interactions. This mechanism can serve as a building block of more complex regulatory architectures in which αCTD promotes transcription through interactions with both DNA and protein activators.

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

scholarly journals Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Bacterial Transcription ◽  
Fluorescence Measurements ◽  
Dimensional Diffusion ◽  
Single Molecule Studies ◽  
Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

scholarly journals Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

2021 ◽  
Author(s):  
Ondrej Belan ◽  
Consuelo Barroso ◽  
Artur Kaczmarczyk ◽  
Roopesh Anand ◽  
Stefania Federico ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nucleation And Growth ◽  
Single Molecule Analysis ◽  
Stimulation Of

scholarly journals Gating Dynamics of the Acetylcholine Receptor Extracellular Domain

2004 ◽  
Vol 123 (4) ◽  
pp. 341-356 ◽  
Author(s):  
Sudha Chakrapani ◽  
Timothy D. Bailey ◽  
Anthony Auerbach
Keyword(s):  
Conformational Change ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Extracellular Domain ◽  
Mouse Muscle ◽  
Α Subunit ◽  
Acetylcholine Binding Protein ◽  
Almost All ◽  
Loop 2 ◽  
Sandwich Core

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

scholarly journals Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

2015 ◽  
Vol 112 (44) ◽  
pp. 13467-13472 ◽  
Author(s):  
Danya J. Martell ◽  
Chandra P. Joshi ◽  
Ahmed Gaballa ◽  
Ace George Santiago ◽  
Tai-Yen Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Dynamic Equilibrium ◽  
Specific Binding ◽  
Binding Mode ◽  
Biased Sampling ◽  
Single Molecule Fret ◽  
Dead End ◽  
The Dead

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ70-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)—a Cu+-responsive MerR-family metalloregulator—modulates RNAP interactions with CueR’s cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopAinteractions. We find that RNAP can form two noninterconverting complexes at PcopAin the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a “biased sampling” instead of “dynamic equilibrium shifting” mechanism in regulating transcription initiation; it modulates RNAP’s binding–unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopAinto its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

2018 ◽  
Vol 115 (50) ◽  
pp. E11604-E11613 ◽  
Author(s):  
Eric A. Galburt
Keyword(s):  
Rate Constants ◽  
Flux Ratio ◽  
Stringent Response ◽  
Kinetic Mechanism ◽  
Regulation Of Transcription ◽  
Sequence Motifs ◽  
Response Factors ◽  
Initiation Rate ◽  
Dna Sequence Motifs ◽  
Kinetics Of

The regulation of transcription allows cells to adjust the rate of RNA polymerases (RNAPs) initiated in a promoter-specific manner. Classically, transcription factors are directed to a subset of promoters via the recognition of DNA sequence motifs. However, a unique class of regulators is recruited directly through interactions with RNAP. Surprisingly, these factors may still possess promoter specificity, and it has been postulated that the same kinetic mechanism leads to different regulatory outcomes depending on a promoter’s basal rate constants. However, mechanistic studies of regulation typically report factor activity in terms of changes in the thermodynamics or kinetics of individual steps or states while qualitatively linking these observations to measured changes in transcript production. Here, I present online calculators that allow for the direct testing of mechanistic hypotheses by calculating the steady-state transcript flux in the presence and absence of a factor as a function of initiation rate constants. By evaluating how the flux ratio of a single kinetic mechanism varies across promoter space, quantitative insights into the potential of a mechanism to generate promoter-specific regulatory outcomes are obtained. Using these calculations, I predict that the mycobacterial transcription factor CarD is capable of repression in addition to its known role as an activator of ribosomal genes. In addition, a modification of the mechanism of the stringent response factors DksA/guanosine 5′-diphosphate 3′-diphosphate (ppGpp) is proposed based on their ability to differentially regulate transcription across promoter space. Overall, I conclude that a multifaceted kinetic mechanism is a requirement for differential regulation by this class of factors.

scholarly journals p53 dynamically directs TFIID assembly on target gene promoters

2016 ◽  
Author(s):  
R. A. Coleman ◽  
Z. Qiao ◽  
S. K. Singh ◽  
C. S. Peng ◽  
M. Cianfrocco ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Gene Networks ◽  
Target Gene ◽  
Core Promoter ◽  
Gene Promoters ◽  
Binding Modes ◽  
Dissociation Kinetics ◽  
Target Promoters

AbstractThe p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

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