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Holoenzymes

Holoenzymes are multisubunit protein complexes that catalyze biological reactions.
They consist of an apoenzyme (the protein portion) and one or more cofactors, such as coenzymes, metal ions, or prosthetic groups, which are essential for the enzyme's activity.
Holoenzymes play crucial roles in various metabolic pathways, gene expression regulation, and signal transduction processes.
Studying the structure, function, and interaction of holoenzyme components is crucial for understanding cellular mechanisms and developing targeted therapies for related diseases.

Most cited protocols related to «Holoenzymes»

1
Purification of Proteasome Subcomplexes
Cited 17 times
Endogenous holoenzyme, core particle 41 (link), and lid subcomplex 42 (link) were purified from S. cerevisiae essentially as described. The base subcomplex was purified according to protocols for the holoenzyme preparation, but with minor modifications as described in the Full Methods. Details of yeast strain construction are provided in Table S1.
Yeast lid was recombinantly expressed from three plasmids in E. coli BL21-star (DE3), and purified on anti-FLAG M2 resin and by size-exclusion chromatography (see Full Methods).
Lander G.C., Estrin E., Matyskiela M.E., Bashore C., Nogales E, & Martin A. (2012). Complete subunit architecture of the proteasome regulatory particle. Nature, 482(7384), 186-191.
Publication 2011
Escherichia coli Holoenzymes Molecular Sieve Chromatography Plasmids Resins, Plant Saccharomyces cerevisiae Strains
2
Competitive Kinetics of ptRNA Substrates
Cited 13 times
ptRNAs and ptRNAMet with randomized leader sequences were produced by in vitro transcription from PCR-generated templates. RNase P processing reactions were performed with 1 μM ptRNA and 5 nM RNase P holoenzyme (equimolar RNase P RNA and C5). Product and unreacted ptRNA were separated by PAGE. cDNA libraries for Illumina sequencing were prepared from unreacted ptRNA at each given timepoint. Primers with degenerate barcodes were used to detect biased PCR amplification of certain sequences. Sequencing was performed on an Illumina GA2. Relative rate constants rk for individual substrate variants were calculated from changes in the distribution of substrates over time, using a multiple turnover reaction scheme for competitive substrate kinetics, which was extended to several thousand substrates. Computational modeling for the rules of substrate discrimination was performed by ordinary least squares regression of the matrix of values for ln(rk) for each sequence variant according to four models of increasing complexity. The quality of the different models was judged by the correlation coefficient between a dataset calculated from values obtained from the regression analysis and the set of experimentally obtained values for ln(rk).
Guenther U.P., Yandek L.E., Niland C.N., Campbell F.E., Anderson D., Anderson V.E., Harris M.E, & Jankowsky E. (2013). Hidden specificity in an apparently non-specific RNA-binding protein. Nature, 502(7471), 385-388.
Publication 2013
cDNA Library Discrimination, Psychology Holoenzymes Kinetics Oligonucleotide Primers RNase P Signal Peptides Transcription, Genetic
3
Tetrahymena Telomerase Purification and Activity
Cited 13 times
Tetrahymena strain constructions and steps of tag-based affinity purification were done as described6 (link) and in Methods. To label the tagged subunit for EM, telomerase particles were first purified using anti-FLAG M2 antibody resin then bound to rabbit-IgG resin. The telomerase-bound IgG resin was then incubated with Fab derived from anti-FLAG M2 IgG, and elution was effected by protease cleavage. Negatively stained EM specimens were prepared with fresh telomerase samples, stained with 0.8% uranyl formate, and examined with an FEI Tecnai F20 electron microscope operated at 200 kV. Frozen hydrated specimens were prepared using Quantifoil grids and imaged with an FEI Titan Krios electron microscope operated at 120 kV. The image processing tasks, including image classification and RCT reconstruction, were performed as described in Methods.
Telomerase activity assays were performed at room temperature using purified telomerase complexes on FLAG antibody resin with standard Tetrahymena holoenzyme reaction conditions using 0.3 μM 32 (link)P-labeled dGTP. Holoenzyme reconstitution used synthetic genes encoding TERT-f, p75, p65, p50, p45, and p19 for expression in RRL; TER purified following in vitro transcription by T7 RNA polymerase; and N-terminally His6-tagged Teb1BC purified following bacterial expression21 (link).
Full Methods and any associated references are available in the online version of the paper.
Jiang J., Miracco E.J., Hong K., Eckert B., Chan H., Cash D.D., Min B., Zhou Z.H., Collins K, & Feigon J. (2013). The architecture of Tetrahymena telomerase holoenzyme. Nature, 496(7444), 187-192.
Publication 2013
Adjustment Disorders anti-IgG Antibodies, Anti-Idiotypic Bacteria bacteriophage T7 RNA polymerase Biological Assay Chromatography, Affinity Cytokinesis deoxyguanosine triphosphate Electron Microscopy Freezing Holoenzymes Immunoglobulins Peptide Hydrolases Protein Subunits Rabbits Reconstructive Surgical Procedures Resins, Plant Strains Synthetic Genes Telomerase TERT protein, human Tetrahymena Transcription, Genetic uranyl formate
4
Competitive Kinetics of ptRNA Substrates
Cited 13 times
ptRNAs and ptRNAMet with randomized leader sequences were produced by in vitro transcription from PCR-generated templates. RNase P processing reactions were performed with 1 μM ptRNA and 5 nM RNase P holoenzyme (equimolar RNase P RNA and C5). Product and unreacted ptRNA were separated by PAGE. cDNA libraries for Illumina sequencing were prepared from unreacted ptRNA at each given timepoint. Primers with degenerate barcodes were used to detect biased PCR amplification of certain sequences. Sequencing was performed on an Illumina GA2. Relative rate constants rk for individual substrate variants were calculated from changes in the distribution of substrates over time, using a multiple turnover reaction scheme for competitive substrate kinetics, which was extended to several thousand substrates. Computational modeling for the rules of substrate discrimination was performed by ordinary least squares regression of the matrix of values for ln(rk) for each sequence variant according to four models of increasing complexity. The quality of the different models was judged by the correlation coefficient between a dataset calculated from values obtained from the regression analysis and the set of experimentally obtained values for ln(rk).
Guenther U.P., Yandek L.E., Niland C.N., Campbell F.E., Anderson D., Anderson V.E., Harris M.E, & Jankowsky E. (2013). Hidden specificity in an apparently non-specific RNA-binding protein. Nature, 502(7471), 385-388.
Publication 2013
cDNA Library Discrimination, Psychology Holoenzymes Kinetics Oligonucleotide Primers RNase P Signal Peptides Transcription, Genetic
5
Structural Determination of RNAP-DNA Complex
Cited 10 times

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Publication 2015
Cell Nucleus DNA, Double-Stranded Electrons Escherichia coli Helix (Snails) Holoenzymes Microtubule-Associated Proteins

Most recents protocols related to «Holoenzymes»

1
Single-round In Vitro Transcription of Riboswitches
All single-round in vitro transcription reactions for TECprobe-ML experiments were performed as 60 μl reactions containing 1X Transcription Buffer [20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA], 0.1 mg/ml Molecular Biology-Grade BSA (Invitrogen), 100 μM high-purity NTPS (Cytiva), 10 nM randomly biotinylated template DNA, and 0.024 U/μl E. coli RNA polymerase holoenzyme (New England Biolabs). Single-round in vitro transcription reactions for TECprobe-SL experiments were performed as 60 μl reactions containing the same reagents as TECprobe-ML experiments except that the reactions used a 10 nM template DNA that contains an internal etheno-dA stall site downstream of a Cbe pfl ZTP riboswitch variant in which the poly-U tract was removed, and either 100 or 500 μM high-purity NTPs, as indicated. Transcription reactions for experiments where DMS was used to probe RNA structures contained additional Tris-HCl (pH 8.0) at final concentration of 100 mM to minimize pH changes during the chemical probing reaction. At the time of preparation, each TECprobe-ML reaction was 48 μl due to the omission of 10X (1 μM) streptavidin (Promega) and 10X Start Solution [100 mM MgCl2, 100 μg/ml rifampicin (Gold Biotechnology)] from the reaction. Each TECprobe-SL reaction was 54 μl due to the omission of 10X Start Solution from the reaction.
The composition of the single-round in vitro transcription master mix varied depending on the riboswitch system that was assessed. In vitro transcription reactions for the Cbe pfl ZTP riboswitch contained 2% (v/v) DMSO and, when present, 1 mM ZMP (Sigma-Aldrich). In vitro transcription reactions for the Bce crcB fluoride riboswitch that were performed in the presence of fluoride contained 10 mM NaF (Sigma-Aldrich). In vitro transcription reactions for the Cba ppGpp riboswitch and C69A ppGpp riboswitch variant contained 500 nM NusA and, when present, 250 μM ppGpp (Guanosine-3’,5’-bisdiphosphate) (Jena Bioscience).
Single-round in vitro transcription reactions were incubated at 37°C for 10 min to form open promoter complexes. For TECprobe-ML reactions, 6 μl of 1 μM streptavidin was then added for a final concentration of 100 nM streptavidin, and reactions were incubated for an additional 10 min at 37°C; TECprobe-SL reactions did not include streptavidin but were still incubated for a total of 20 min at 37°C. Transcription was initiated by adding 6 μl of 10X Start Solution to the reaction for a final concentration of 10 mM MgCl2 and 10 μg/ml rifampicin. The transcription reaction was incubated at 37°C for 2 min before chemical probing was performed as described below in the section RNA chemical probing.
Szyjka C.E, & Strobel E.J. (2023). Observation of coordinated cotranscriptional RNA folding events. bioRxiv.
Publication Preprint 2023
Buffers Dithiothreitol DNA, A-Form DNA-Directed RNA Polymerase Edetic Acid Escherichia coli Fluorides Gold Guanosine Guanosine Tetraphosphate Holoenzymes Magnesium Chloride Poly U Promega Riboswitch Rifampin Streptavidin Sulfoxide, Dimethyl Transcription, Genetic Tromethamine
2
Enzymatic Tools for Molecular Biology
Q5 High-Fidelity DNA Polymerase, Vent (exo-) DNA polymerase, Sulfolobus DNA Polymerase IV, E. coli RNA Polymerase holoenzyme, Mth RNA Ligase (as part of the 5’ DNA Adenylation kit), T4 RNA Ligase 2 truncated KQ, ET SSB, RNase H, and RNase If were purchased from New England Biolabs. TURBO DNase, SuperaseIN, SuperScript II, SuperScript III, and BSA were purchased from ThermoFisher. Streptavidin was purchased from Promega. NusA was a gift from J. Roberts (Cornell University)
Szyjka C.E, & Strobel E.J. (2023). Observation of coordinated cotranscriptional RNA folding events. bioRxiv.
Publication Preprint 2023
Deoxyribonucleases DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase DNA Polymerase beta Endoribonucleases Escherichia coli Holoenzymes Promega Ribonuclease H RNA Ligase (ATP) Streptavidin Sulfolobus
3
Primer Extension Assays for DNA Polymerases
Primer extension reactions (25 μl) were based on method in (33 (link),34 (link)), utilizing purified human Pol δ (40 nM) in a holoenzyme complex with PCNA (40 nM), RFC (10 nM) and RPA (320 nM). RPA, PCNA and RFC were pre-mixed for 10 min on ice in the reaction buffer containing DNA substrate (82.5 ng M13-primer DNA/reaction) and dNTPs (200 μM), before adding Pol δ to start the reactions. Reactions were for 30 min at 37°C before adding 2 μl of STOP solution (50 mM Tris–HCl pH 8.0, 100 mM EDTA, 0.1% w/v SDS and 5 mg/ml of proteinase K). Cy5-labelled products after electrophoresis through a 0.8% agarose TBE gel were visualised using a Typhoon scanner, and unlabelled plasmid DNA was visualised by ethidium bromide staining and placing the gel on a UV transilluminator.
Primer extension reactions by isolated Pol δ complex (POLD1-D4) were in 20 μl containing substrate DNA (21 nt primer oligo annealed to a 70 nt oligo template, each at 15 nM), 10 mM MgCl2, 40 mM Tris–HCl pH7.5, 1 mM DTT, 0.2 mg/ml BSA, 50 mM NaCl and 200 μM of each dNTP. Primer extension assays using human DNA polymerase η and polymerase k were in buffer 40 mM Tris–HCl pH 8.0, 10 mM DTT, 0.25 mg/ml BSA, 60 mM KCl, 2.5% glycerol, 5 mM MgCl2, 200 μM dNTPs. Primer extension by E. coli polymerase III core (DnaE) and polymerase I were in buffer 10 mM magnesium acetate, 40 mM HEPES–NaOH pH 8.0, 0.1 mg/ml BSA, 10 mM DTT and 200 μM dNTPs. Unless stated otherwise, all polymerase primer extension assays were for 30 min at 37°C after adding DNA. Reactions were halted by adding 5 μl of stock STOP solution. Stopped reactions were mixed with loading dye (20% glycerol, 78% formamide and Orange G) for electrophoresis through 10% acrylamide (19:1 acrylamide: bis-acrylamide) TBE denaturing (8 M urea) gels, at 5 W for 3 h. Primer extension products were visualised via the Cy5-DNA end label using a Typhoon scanner, and files were quantified using ImageJ and Prism software. For primer extension reactions containing HelQ, proteins were pre-mixed in their storage buffers, in isolation from reaction buffer and DNA, and reactions commenced by adding buffer containing DNA and dNTPs.
He L., Lever R., Cubbon A., Tehseen M., Jenkins T., Nottingham A.O., Horton A., Betts H., Fisher M., Hamdan S.M., Soultanas P, & Bolt E.L. (2023). Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing. Nucleic Acids Research, 51(4), 1740-1749.
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Publication 2023
Acrylamide Biological Assay Buffers Edetic Acid Electrophoresis Endopeptidase K Escherichia coli Ethidium Bromide formamide Gels Glycerin HEL308 protein, human HEPES Holoenzymes Homo sapiens isolation magnesium acetate Magnesium Chloride Oligonucleotide Primers Oligonucleotides Orange G Plasmids POLD1 protein, human prisma Proliferating Cell Nuclear Antigen Proteins Rad30 protein Sepharose Sodium Chloride Tromethamine Typhoons Urea
4
Measuring Polymerase Pause Dynamics
The intrinsic flexibility of ssDNA together with the slow average strand displacement rates of the mitochondrial holoenzymes used in this work hindered the accurate identification of pause events (8 (link)). Nevertheless, identification of pause-free velocities (V(f)) allowed us to calculate the average residence time at the pause state per nucleotide at each tension, , as the difference between the average total residence time per nucleotide (  = 1/ Vmean(f)) and the residence time in the active state (  = 1/ V(f)). The tension dependencies of of each polymerase under study were fitted with Eq. 1.
Plaza-G.A. I., Lemishko K.M., Crespo R., Truong T.Q., Kaguni L.S., Cao-García F.J., Ciesielski G.L, & Ibarra B. (2023). Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB. Nucleic Acids Research, 51(4), 1750-1765.
Publication 2023
DNA, Single-Stranded Holoenzymes Mitochondria Nucleotides
5
Processive Replication Dynamics
The average replication rate at each tension (Vmean(f)) was determined by a line fit to the traces showing the number of replicated nucleotides versus time. The final average rate at each tension was obtained by averaging over all of the traces taken within similar tension values (±0.5 pN). Average replication rate without pauses at each tension (pause-free velocity, V(f)) was determined with an algorithm that computes the instantaneous velocities of the trajectory, averaging the position of the holoenzyme along the DNA over sliding time windows, as described previously (8 (link)). Tension dependent pause-free velocities were fitted to the strand displacement model described in SI and (52 (link)).
Plaza-G.A. I., Lemishko K.M., Crespo R., Truong T.Q., Kaguni L.S., Cao-García F.J., Ciesielski G.L, & Ibarra B. (2023). Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB. Nucleic Acids Research, 51(4), 1750-1765.
Publication 2023
DNA Replication Holoenzymes Nucleotides

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1
Ni nta agarose by Qiagen
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Ni-NTA agarose is a solid-phase affinity chromatography resin designed for the purification of recombinant proteins containing a histidine-tag. It consists of nickel-nitrilotriacetic acid (Ni-NTA) coupled to agarose beads, which selectively bind to the histidine-tagged proteins.
2
E coli rnap holoenzyme by New England Biolabs
E. coli RNAP holoenzyme is a core component of the bacterial transcription machinery. It is a multi-subunit complex responsible for the transcription of genetic information from DNA to RNA.
3
E coli rna polymerase holoenzyme by New England Biolabs
Sourced in United States
E. coli RNA polymerase holoenzyme is a multi-subunit enzyme that is responsible for the transcription of DNA into RNA in Escherichia coli. It consists of a core enzyme composed of five subunits (2α, 1β, 1β', 1ω) and a sigma factor that confers promoter specificity. The holoenzyme is essential for the initiation of transcription and the subsequent elongation and termination of RNA synthesis.
4
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Superdex 200 is a size-exclusion chromatography medium used for the separation and purification of proteins, peptides, and other biomolecules. It is composed of highly cross-linked agarose beads that allow for efficient separation based on molecular size. The Superdex 200 matrix provides a wide fractionation range and high resolution, making it a versatile tool for a variety of applications in biotechnology and life science research.
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E coli rna polymerase holoenzyme by Illumina
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The E. coli RNA polymerase holoenzyme is a laboratory equipment product that serves as a core component in the transcription process. It is responsible for the synthesis of RNA molecules from a DNA template, which is a fundamental step in gene expression. The holoenzyme consists of the RNA polymerase core enzyme and a sigma factor, which together initiate and regulate the transcription of genetic information.
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More about "Holoenzymes"

Holoenzymes are complex, multi-subunit protein structures that play a crucial role in catalyzing biological reactions.
They are composed of an apoenzyme (the protein portion) and one or more essential cofactors, such as coenzymes, metal ions, or prosthetic groups.
These cofactors are necessary for the enzyme's activity and function.
Holoenzymes are involved in a wide range of cellular processes, including metabolic pathways, gene expression regulation, and signal transduction.
Understanding the structure, function, and interactions of holoenzyme components is vital for researchers studying cellular mechanisms and developing targeted therapies for related diseases.
Techniques like Ni-NTA agarose, a nickel-nitrilotriacetic acid resin used for protein purification, are often employed in holoenzyme research.
The E. coli RNA polymerase (RNAP) holoenzyme, a key player in bacterial transcription, is a well-studied holoenzyme system.
Researchers may also utilize size-exclusion chromatography techniques, such as Superdex 200, to separate and analyze holoenzyme complexes.
Sample preparation methods, such as the Vitrobot Mark IV, a device for cryo-electron microscopy (cryo-EM) sample preparation, can be employed to study the structure of holoenzymes.
Data analysis software like Prism 6 and imaging tools like the FLA-5000 PhosphorImager can also be used to support holoenzyme research.
Radioactive labeling techniques, such as the incorporation of [α-32P]UTP, can aid in the detection and analysis of holoenzyme components and their interactions.
Additionally, computational tools like SigmaPlot can be utilized for data visualization and statistical analysis in holoenzyme studies.
By leveraging these techniques and tools, researchers can gain deeper insights into the complex world of holoenzymes and their crucial roles in cellular processes.
This knowledge can ultimately lead to the development of more effective targeted therapies for related diseases.