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 Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

2019 ◽  
Author(s):  
Zhu Liu ◽  
Xu Dong ◽  
Hua-Wei Yi ◽  
Ju Yang ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complex Structure ◽  
Binding Mode ◽  
Structural Basis ◽  
Ubiquitinated Proteins ◽  
Single Molecule Fret ◽  
Compact State ◽  
A Charge ◽  
Substrate Proteins ◽  
Ubiquitinated Substrate

ABSTRACTThe interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, and it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among three distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have solved the complex structure between Rpn13NTD and K48-diUb. In the structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge reversal mutation in Rpn13NTD can weaken the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, blockage of the access of the distal Ub to Rpn13NTD with a proximity attached Ub monomer can also disrupt the interaction between Rpn13 and K48-diUb. Together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

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 Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

2015 ◽  
Vol 43 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Namita Bisaria ◽  
Daniel Herschlag
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Specific Interaction ◽  
Monovalent Cations ◽  
Folding Kinetics ◽  
Rna Molecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

scholarly journals Single Molecule FRET Characterization of Structural Changes in Antibodies Induced by Enzymatic Deglycosylation

2014 ◽  
Vol 106 (2) ◽  
pp. 49a
Author(s):  
Mark S. Piraino ◽  
Michael T. Kelliher ◽  
Ramiah D. Jacks ◽  
Madeline E. Gemoules ◽  
Jihad Aburas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Single Molecule Fret

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

scholarly journals Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly

2019 ◽  
Vol 47 (20) ◽  
pp. 10842-10851 ◽  
Author(s):  
David H Beier ◽  
Tucker J Carrocci ◽  
Clarisse van der Feltz ◽  
U Sandy Tretbar ◽  
Joshua C Paulson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Conformational Dynamics ◽  
Spliceosome Assembly ◽  
Single Molecule Fret ◽  
Dead Box ◽  
U2 Snrnp ◽  
The Dead

Abstract The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

scholarly journals DNA-sequence dependent positioning of the linker histone in a chromatosome: a single-pair FRET study

2020 ◽  
Author(s):  
Madhura De ◽  
Mehmet Ali Oeztuerk ◽  
Katalin Toth ◽  
Rebecca C. Wade
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Binding Mode ◽  
Linker Histone ◽  
Single Pair ◽  
Globular Domain ◽  
Binding Modes ◽  
Single Molecule Fret ◽  
Fret Efficiency ◽  
Order Structures

The linker histone (LH) associates with the nucleosome with its globular domain (gH) binding in an on or off-dyad binding mode. The positioning of the LH may play a role in the compaction of higher-order structures of chromatin. Preference for different binding modes has been attributed to the LHs amino acid sequence. We here study the effect of the linker DNA (L-DNA) sequence on the positioning of a full-length LH, Xenopus laevis H1.0b, by employing single-molecule FRET spectroscopy. Chromatosomes were fluorescently labelled on one of the two 40bp long L-DNA arms, and on the gH. We varied 11bp of DNA flanking the core (non-palindromic Widom 601) of each chromatosome construct, making them either A-tract, purely GC, or mixed, with 64% AT. The gH consistently exhibited higher FRET efficiency with the L-DNA containing the A-tract, than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA, compared to the pure-GC stretch. We explain our observations with a FRET-distance restrained model that shows that the gH binds on-dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove.

scholarly journals The many states of STIM1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marc Fahrner ◽  
Christoph Romanin
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Single Molecule Fret ◽  
A Cell ◽  
The Many ◽  
Fine Tune

Harnessing single-molecule FRET illuminates the structural changes necessary for a protein to fine-tune the influx of calcium when reserves inside a cell run low.

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