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Article ; Online: Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

Strobel, Eric J

2023 Volume 691, Page(s) 223–250

Abstract: The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long ... ...

Abstract	The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long and complex transcripts, we developed a procedure for isolating roadblocked E. coli TECs from an in vitro transcription reaction using solid-phase photoreversible immobilization. Our approach uses a modified DNA template that contains both a 5' photocleavable biotin tag and an internal biotin-TEG transcription stall site. Because the footprint of a TEC at the stall site sequesters the biotin-TEG tag, DNA template molecules that contain a TEC can be reversibly immobilized on streptavidin-coated magnetic beads by the 5' photocleavable biotin tag. In contrast, DNA template molecules that do not contain a TEC are retained on the beads because the biotin-TEG tag is exposed and can bind streptavidin. In this way, DNA template molecules that contain a TEC are gently separated from free DNA and DNA that contains non-productive transcription complexes. This procedure yields precisely positioned TECs that are >95% pure with >30% yield relative to the amount of input DNA template. The resulting complexes are >99% stable for at least 3 h and can be used for biochemical investigations of nascent RNA structure and function in the context of E. coli RNAP. The procedure is likely generalizable to any RNAP that arrests at and sequesters the internal biotin-TEG stall site.
MeSH term(s)	Transcription, Genetic ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Streptavidin/genetics ; Biotin/metabolism ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; RNA/chemistry ; DNA/metabolism
Chemical Substances	Streptavidin (9013-20-1) ; Biotin (6SO6U10H04) ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; RNA (63231-63-0) ; DNA (9007-49-2)
Language	English
Publishing date	2023-04-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2023.03.019
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking.

Strobel, Eric J

Bio-protocol

2021 Volume 11, Issue 17, Page(s) e4141

Abstract: Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting ... ...

Abstract	Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription "roadblocking" approaches halt transcription elongation using a protein blockade that is non-covalently attached to the template DNA. I previously developed a strategy for halting
Language	English
Publishing date	2021-09-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325 ; 2331-8325
ISSN (online)	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.4141
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Article: Systematic analysis of cotranscriptional RNA folding using transcription elongation complex display.

Kelly, Skyler L / Strobel, Eric J

bioRxiv : the preprint server for biology

2023

Abstract: RNA can fold into structures that mediate diverse cellular functions. Understanding how RNA primary sequence directs the formation of functional structures requires methods that can comprehensively assess how changes in an RNA sequence affect its ... ...

Abstract	RNA can fold into structures that mediate diverse cellular functions. Understanding how RNA primary sequence directs the formation of functional structures requires methods that can comprehensively assess how changes in an RNA sequence affect its structure and function. Here we have developed a platform for performing high-throughput cotranscriptional RNA biochemical assays, called Transcription Elongation Complex display (TECdisplay). TECdisplay measures RNA function by fractionating a TEC library based on the activity of cotranscriptionally displayed nascent RNA. In this way, RNA function is measured as the distribution of template DNA molecules between fractions of the transcription reaction. This approach circumvents typical RNA sequencing library preparation steps that can cause technical bias. We used TECdisplay to characterize the transcription antitermination activity of 32,768 variants of the
Language	English
Publishing date	2023-12-23
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.12.22.573115
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Article ; Online: Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads.

Strobel, Eric J

The Journal of biological chemistry

2021 Volume 297, Issue 1, Page(s) 100812

Abstract: In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic ... ...

Abstract	In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin-triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.
MeSH term(s)	Base Pairing ; Biotin/chemistry ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/enzymology ; Escherichia coli/genetics ; Light ; Magnetic Phenomena ; Microspheres ; Promoter Regions, Genetic ; RNA/chemistry ; Streptavidin/chemistry ; Transcription Elongation, Genetic
Chemical Substances	RNA (63231-63-0) ; Biotin (6SO6U10H04) ; Streptavidin (9013-20-1) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2021-05-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2021.100812
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Article ; Online: Efficient Linear dsDNA Tagging Using Deoxyuridine Excision*.

Strobel, Eric J

Chembiochem : a European journal of chemical biology

2021 Volume 22, Issue 22, Page(s) 3214–3224

Abstract: Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. ... ...

Abstract	Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3' overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5' overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.
MeSH term(s)	Cloning, Molecular ; DNA/genetics ; DNA/metabolism ; Deoxyuridine/metabolism ; Gene Library ; Oligonucleotides/genetics ; Oligonucleotides/metabolism
Chemical Substances	Oligonucleotides ; DNA (9007-49-2) ; Deoxyuridine (W78I7AY22C)
Language	English
Publishing date	2021-10-01
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2020469-3
ISSN	1439-7633 ; 1439-4227
ISSN (online)	1439-7633
ISSN	1439-4227
DOI	10.1002/cbic.202100425
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Article: Observation of coordinated cotranscriptional RNA folding events.

Szyjka, Courtney E / Strobel, Eric J

bioRxiv : the preprint server for biology

2023

Abstract: RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for ... ...

Abstract	RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from RNA polymerase. Here, we have developed a concise, high-resolution cotranscriptional RNA chemical probing procedure called Transcription Elongation Complex RNA structure probing-Multilength (TECprobe-ML). We validated TECprobe-ML by replicating and extending previous analyses of ZTP and fluoride riboswitch folding, and mapped the folding pathway of a ppGpp-sensing riboswitch. In each system, TECprobe-ML identified coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-ML as an accessible method for mapping cotranscriptional RNA folding pathways.
Language	English
Publishing date	2023-02-21
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.02.21.529405
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Article ; Online: Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

Szyjka, Courtney E / Strobel, Eric J

Nature communications

2023 Volume 14, Issue 1, Page(s) 7839

Abstract	RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.
MeSH term(s)	RNA Folding ; RNA/genetics ; RNA/chemistry ; Nucleic Acid Conformation ; Riboswitch/genetics ; Transcription, Genetic ; DNA-Directed RNA Polymerases/genetics
Chemical Substances	RNA (63231-63-0) ; Riboswitch ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2023-11-29
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-43395-9
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Article ; Online: Cotranscriptional RNA Chemical Probing.

Szyjka, Courtney E / Strobel, Eric J

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2518, Page(s) 291–330

Abstract: Cotranscriptional folding is a fundamental step in RNA biogenesis and the basis for many RNA-mediated gene regulation systems. Understanding how RNA folds as it is synthesized requires experimental methods that can systematically identify intermediate ... ...

Abstract	Cotranscriptional folding is a fundamental step in RNA biogenesis and the basis for many RNA-mediated gene regulation systems. Understanding how RNA folds as it is synthesized requires experimental methods that can systematically identify intermediate RNA structures that form during transcription. Cotranscriptional RNA chemical probing experiments achieve this by applying high-throughput RNA structure probing to an in vitro transcribed array of cotranscriptionally folded intermediate transcripts. In this chapter, we present guidelines and procedures for integrating single-round in vitro transcription using E. coli RNA polymerase with high-throughput RNA chemical probing workflows. We provide an overview of key concepts including DNA template design, transcription roadblocking strategies, single-round in vitro transcription with E. coli RNA polymerase, and RNA chemical probing and describe procedures for DNA template preparation, cotranscriptional RNA chemical probing, RNA purification, and 3' adapter ligation. The end result of these procedures is a purified RNA library that can be prepared for Illumina sequencing using established high-throughput RNA structure probing library construction strategies.
MeSH term(s)	DNA ; DNA-Directed RNA Polymerases/chemistry ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli/chemistry ; Escherichia coli/genetics ; RNA/chemistry ; RNA/genetics ; RNA Folding ; RNA Probes ; Sequence Analysis, RNA ; Transcription, Genetic
Chemical Substances	RNA Probes ; RNA (63231-63-0) ; DNA (9007-49-2) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2022-06-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2421-0_17
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing

Courtney E. Szyjka / Eric J. Strobel

Nature Communications, Vol 14, Iss 1, Pp 1-

2023 Volume 22

Abstract: Abstract RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for ... ...

Abstract	Abstract RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.
Keywords	Science ; Q
Subject code	612
Language	English
Publishing date	2023-11-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
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Article ; Online: Isolation of synchronized E. coli elongation complexes for solid-phase and solution-based in vitro transcription assays.

Strobel, Eric J / Kelly, Skyler L / Szyjka, Courtney E

Methods in enzymology

2022 Volume 675, Page(s) 159–192

Abstract: Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. ... ...

Abstract	Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. coli RNAP TECs from an in vitro transcription reaction. Our system uses a custom 5' leader sequence, called C3-SC1 to immobilize synchronized TECs on magnetic beads so that free DNA and non-productive transcription complexes can be depleted. The synchronized elongation complexes isolated by our procedure, called
MeSH term(s)	DNA/chemistry ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; RNA/chemistry ; Transcription, Genetic
Chemical Substances	RNA (63231-63-0) ; DNA (9007-49-2) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2022-09-09
Publishing country	United States
Document type	Journal Article
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2022.07.008
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