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Article ; Online: Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by

Sreenivasan, Raashi / Shkel, Irina A / Chhabra, Munish / Drennan, Amanda / Heitkamp, Sara / Wang, Hao-Che / Sridevi, Malavika A / Plaskon, Dylan / McNerney, Christina / Callies, Katelyn / Cimperman, Clare K / Record, M Thomas

Biochemistry

2020 Volume 59, Issue 16, Page(s) 1565–1581

Abstract: FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of ... ...

Abstract	FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λP
MeSH term(s)	Carbocyanines/chemistry ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/enzymology ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/metabolism ; Fluorescence ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes/chemistry ; Kinetics ; Nucleic Acid Conformation ; Promoter Regions, Genetic ; Protein Binding
Chemical Substances	Carbocyanines ; Escherichia coli Proteins ; Fluorescent Dyes ; cyanine dye 3 ; cyanine dye 5 ; DNA (9007-49-2) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2020-04-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.0c00098
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Article: Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Biochemistry. 2020 Mar. 27, v. 59, no. 16

2020

Abstract: FRET (fluorescence resonance energy transfer) between far-upstream (−100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC ... ...

Abstract	FRET (fluorescence resonance energy transfer) between far-upstream (−100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with −100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I₁E), mid (I₁M), and late (I₁L)], followed by conversion of {CC} to OC via I₁L. FRET and PIFE are first observed for I₁E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I₁E, decreasing the Cy3−Cy5 distance to ∼75 Å and making RNAP–DNA contacts at −100 and +14. We propose that far-upstream DNA wraps on the upper β′-clamp while downstream DNA contacts the top of the β-pincer in I₁E. Converting I₁E to I₁M (∼1 s time scale) reduces FRET efficiency with little change in −100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on β′) away from downstream DNA (on β) to increase the Cy3−Cy5 distance by ∼14 Å. FRET increases greatly in converting I₁M to I₁L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3−Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I₁L is converted to the initial unstable OC (I₂). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.
Keywords	DNA ; DNA-directed RNA polymerase ; Escherichia coli ; energy transfer ; fluorescence ; isomerization
Language	English
Dates of publication	2020-0327
Size	p. 1565-1581.
Publishing place	American Chemical Society
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.0c00098
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Article ; Online: HORI

Sreenivasan Raashi / Shameer Khader / Sundaramurthy Pandurangan / Gakkhar Sunita / Sowdhamini Ramanathan

BMC Bioinformatics, Vol 11, Iss Suppl 1, p S

a web server to compute Higher Order Residue Interactions in protein structures

2010 Volume 24

Abstract: Abstract Background Folding of a protein into its three dimensional structure is influenced by both local and global interactions within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones, play a vital role in ... ...

Abstract	Abstract Background Folding of a protein into its three dimensional structure is influenced by both local and global interactions within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones, play a vital role in attaining the stable conformation of the protein structure. It is generally agreed that higher order interactions make significant contribution to the potential energy landscape of folded proteins and therefore it is important to identify them to estimate their contributions to overall stability of a protein structure. Results We developed HORI [Higher order residue interactions in proteins], a web server for the calculation of global and local higher order interactions in protein structures. The basic algorithm of HORI is designed based on the classical concept of four-body nearest-neighbour propensities of amino-acid residues. It has been proved that higher order residue interactions up to the level of quadruple interactions plays a major role in the three-dimensional structure of proteins and is an important feature that can be used in protein structure analysis. Conclusion HORI server will be a useful resource for the structural bioinformatics community to perform analysis on protein structures based on higher order residue interactions. HORI server is a highly interactive web server designed in three modules that enables the user to analyse higher order residue interactions in protein structures. HORI server is available from the URL: http://caps.ncbs.res.in/hori
Keywords	Biology (General) ; QH301-705.5 ; Science ; Q ; DOAJ:Biology ; DOAJ:Biology and Life Sciences
Subject code	612
Language	English
Publishing date	2010-01-01T00:00:00Z
Publisher	BioMed Central
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins.

Jarmoskaite, Inga / Denny, Sarah K / Vaidyanathan, Pavanapuresan P / Becker, Winston R / Andreasson, Johan O L / Layton, Curtis J / Kappel, Kalli / Shivashankar, Varun / Sreenivasan, Raashi / Das, Rhiju / Greenleaf, William J / Herschlag, Daniel

Molecular cell

2019 Volume 74, Issue 5, Page(s) 966–981.e18

Abstract: High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for ... ...

Abstract	High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.
MeSH term(s)	Amino Acid Sequence/genetics ; Humans ; Kinetics ; Nucleic Acid Conformation ; Protein Binding/genetics ; RNA, Messenger/genetics ; RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/genetics ; Ribosomes/chemistry ; Ribosomes/genetics
Chemical Substances	PUM1 protein, human ; PUM2 protein, human ; RNA, Messenger ; RNA-Binding Proteins
Language	English
Publishing date	2019-05-08
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2019.04.012
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Article: A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins

Jarmoskaite, Inga / Denny, Sarah K / Vaidyanathan, Pavanapuresan P / Becker, Winston R / Andreasson, Johan O.L / Layton, Curtis J / Kappel, Kalli / Shivashankar, Varun / Sreenivasan, Raashi / Das, Rhiju / Greenleaf, William J / Herschlag, Daniel

Molecular cell. 2019 June 06, v. 74, no. 5

2019

Abstract	High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.
Keywords	RNA ; RNA-binding proteins ; crosslinking ; humans ; quantitative analysis ; ribosomes ; thermodynamic models ; thermodynamics
Language	English
Dates of publication	2019-0606
Size	p. 966-981.e18.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2019.04.012
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Article ; Online: HORIBALFRE program: Higher Order Residue Interactions Based ALgorithm for Fold REcognition.

Sundaramurthy, Pandurangan / Sreenivasan, Raashi / Shameer, Khader / Gakkhar, Sunita / Sowdhamini, Ramanathan

Bioinformation

2011 Volume 7, Issue 7, Page(s) 352–359

Abstract: Understanding the functional and structural implication of a protein encoded in novel genes using function association or fold recognition approaches remains to be a challenging task in the current era of genomes, metagenomes and personal genomes. In an ... ...

Abstract	Understanding the functional and structural implication of a protein encoded in novel genes using function association or fold recognition approaches remains to be a challenging task in the current era of genomes, metagenomes and personal genomes. In an attempt to enhance potential-based fold-recognition methods in recognizing remote homology between proteins, we propose a new approach "Higher Order Residue Interaction Based ALgorithm for Fold REcognition (HORIBALFRE)". Higher order residue interactions refer to a class of interactions in protein structures mediated by C(α) or C(β) atoms within a pre-defined distance cut-off. Higher order residue interactions (pairwise, triplet and quadruplet interactions) play a vital role in attaining the stable conformation of a protein structure. In HORIBALFRE, we incorporated the potential contributions from two body (pairwise) interactions, three body (triplet interactions) and four-body (quadruple interaction) interactions, to implement a new fold recognition algorithm. Core of HORIBALFRE algorithm includes the potentials generated from a library of protein structure derived from manually curated CAMPASS database of structure based sequence alignment. We used Fischer's dataset, with 68 templates and 56 target sequences, derived from SCOP database and performed one-against-all sequence alignment using TCoffee. Various potentials were derived using custom scripts and these potentials were incorporated in the HORIBALFRE algorithm. In this manuscript, we report outline of a novel fold recognition algorithm and initial results. Our results show that inclusion of quadruplet class of higher order residue interaction improves fold recognition.
Language	English
Publishing date	2011-12-10
Publishing country	Singapore
Document type	Journal Article
ZDB-ID	2203786-X
ISSN	0973-2063 ; 0973-2063
ISSN (online)	0973-2063
ISSN	0973-2063
DOI	10.6026/97320630007352
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Article ; Online: HORI: a web server to compute Higher Order Residue Interactions in protein structures.

Sundaramurthy, Pandurangan / Shameer, Khader / Sreenivasan, Raashi / Gakkhar, Sunita / Sowdhamini, Ramanathan

BMC bioinformatics

2010 Volume 11 Suppl 1, Page(s) S24

Abstract: Background: Folding of a protein into its three dimensional structure is influenced by both local and global interactions within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones, play a vital role in attaining the ...

Abstract	Background: Folding of a protein into its three dimensional structure is influenced by both local and global interactions within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones, play a vital role in attaining the stable conformation of the protein structure. It is generally agreed that higher order interactions make significant contribution to the potential energy landscape of folded proteins and therefore it is important to identify them to estimate their contributions to overall stability of a protein structure. Results: We developed HORI [Higher order residue interactions in proteins], a web server for the calculation of global and local higher order interactions in protein structures. The basic algorithm of HORI is designed based on the classical concept of four-body nearest-neighbour propensities of amino-acid residues. It has been proved that higher order residue interactions up to the level of quadruple interactions plays a major role in the three-dimensional structure of proteins and is an important feature that can be used in protein structure analysis. Conclusion: HORI server will be a useful resource for the structural bioinformatics community to perform analysis on protein structures based on higher order residue interactions. HORI server is a highly interactive web server designed in three modules that enables the user to analyse higher order residue interactions in protein structures. HORI server is available from the URL: http://caps.ncbs.res.in/hori.
MeSH term(s)	Databases, Protein ; Internet ; Models, Molecular ; Protein Conformation ; Protein Folding ; Proteins/chemistry ; Software ; User-Computer Interface
Chemical Substances	Proteins
Language	English
Publishing date	2010-01-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2041484-5
ISSN	1471-2105 ; 1471-2105
ISSN (online)	1471-2105
ISSN	1471-2105
DOI	10.1186/1471-2105-11-S1-S24
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Article ; Online: A Potent and Selective PARP11 Inhibitor Suggests Coupling between Cellular Localization and Catalytic Activity.

Kirby, Ilsa T / Kojic, Ana / Arnold, Moriah R / Thorsell, Ann-Gerd / Karlberg, Tobias / Vermehren-Schmaedick, Anke / Sreenivasan, Raashi / Schultz, Carsten / Schüler, Herwig / Cohen, Michael S

Cell chemical biology

2018 Volume 25, Issue 12, Page(s) 1547–1553.e12

Abstract: Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors ...

Abstract	Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors for these PARPs. There has been comparatively little success in developing selective small-molecule inhibitors of PARPs that catalyze mono-ADP-ribosylation (MARylation), limiting our understanding of the cellular role of MARylation. Here we describe the structure-guided design of inhibitors of PARPs that catalyze MARylation. The most selective analog, ITK7, potently inhibits the MARylation activity of PARP11, a nuclear envelope-localized PARP. ITK7 is greater than 200-fold selective over other PARP family members. Using live-cell imaging, we show that ITK7 causes PARP11 to dissociate from the nuclear envelope. These results suggest that the cellular localization of PARP11 is regulated by its catalytic activity.
MeSH term(s)	Biocatalysis/drug effects ; HeLa Cells ; Humans ; Molecular Structure ; Poly(ADP-ribose) Polymerase Inhibitors/chemical synthesis ; Poly(ADP-ribose) Polymerase Inhibitors/chemistry ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Poly(ADP-ribose) Polymerases/metabolism ; Protein Transport/drug effects ; Quinazolinones/chemical synthesis ; Quinazolinones/chemistry ; Quinazolinones/pharmacology
Chemical Substances	Poly(ADP-ribose) Polymerase Inhibitors ; Quinazolinones ; Poly(ADP-ribose) Polymerases (EC 2.4.2.30)
Language	English
Publishing date	2018-10-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2451-9448
ISSN (online)	2451-9448
DOI	10.1016/j.chembiol.2018.09.011
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Article: Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA PolymeraseâÎ»PR Promoter Complexes

Sreenivasan, Raashi / Heitkamp Sara / Chhabra Munish / Saecker Ruth / Lingeman Emily / Poulos Mikaela / McCaslin Darrell / Capp Michael W / Artsimovitch Irina / Record M. Thomas

Biochemistry. 2016 Apr. 12, v. 55, no. 14

2016

Abstract: Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40â50 bp of DNA immediately upstream of the â35 region. Kinetic studies demonstrated ... ...

Abstract	Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40â50 bp of DNA immediately upstream of the â35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of â¼13 bp (â11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (â60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAPâÎ»PR CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (â100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 Â± 0.07) and the OC (0.32 Â± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions â100 and +14 on promoter DNA from >300 to <100 Ã. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.
Keywords	DNA ; DNA-directed RNA polymerase ; Escherichia coli ; RNA ; active sites ; atomic force microscopy ; dyes ; energy transfer ; fluorescence ; melting ; models
Language	English
Dates of publication	2016-0412
Size	p. 2174-2186.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021%2Facs.biochem.6b00125
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Article ; Online: Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA Polymerase-λP(R) Promoter Complexes.

Sreenivasan, Raashi / Heitkamp, Sara / Chhabra, Munish / Saecker, Ruth / Lingeman, Emily / Poulos, Mikaela / McCaslin, Darrell / Capp, Michael W / Artsimovitch, Irina / Record, M Thomas

Biochemistry

2016 Volume 55, Issue 14, Page(s) 2174–2186

Abstract: Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that ... ...

Abstract	Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.
MeSH term(s)	Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Bacteriophage lambda/metabolism ; Catalytic Domain ; DNA, Viral/chemistry ; DNA, Viral/metabolism ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/enzymology ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes/chemistry ; Holoenzymes/chemistry ; Holoenzymes/genetics ; Holoenzymes/metabolism ; Kinetics ; Models, Molecular ; Molecular Conformation ; Promoter Regions, Genetic ; Protein Stability ; Protein Subunits/chemistry ; Protein Subunits/genetics ; Protein Subunits/metabolism ; Protein Unfolding ; Recombinant Proteins/chemistry ; Recombinant Proteins/metabolism ; Structural Homology, Protein ; Thermus thermophilus/enzymology
Chemical Substances	Bacterial Proteins ; DNA, Viral ; Escherichia coli Proteins ; Fluorescent Dyes ; Holoenzymes ; Protein Subunits ; Recombinant Proteins ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2016-04-12
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.6b00125
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