This presentation talks about 3 ways to detect protein DNA interactions - Chromatin immunoprecipitation (ChIP), yeast one system (Y1H), and some In-silico tools, in brief. Animations might not work.
''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This assessed presentation was delivered by me, together with other three course mates. The aim of the presentation was to describe the basic principles, methods involved in EMSA, and some of its application in molecular biology to study the interactions between proteins and DNA. Delivered on 9th December, 2013 with Lolomari Songo, Nicholas Leach & Abhay Jethwani.
''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This assessed presentation was delivered by me, together with other three course mates. The aim of the presentation was to describe the basic principles, methods involved in EMSA, and some of its application in molecular biology to study the interactions between proteins and DNA. Delivered on 9th December, 2013 with Lolomari Songo, Nicholas Leach & Abhay Jethwani.
Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
Molecular marker General introduction by K. K. SAHU Sir.KAUSHAL SAHU
Introduction
Molecular marker
Characterstics of molecular marker
Types of molecular marker
. Non PCR Based
. PCR Based
RFLP
RAPD
AFLP
SSR
SNP
Conclusion
References
Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
Molecular marker General introduction by K. K. SAHU Sir.KAUSHAL SAHU
Introduction
Molecular marker
Characterstics of molecular marker
Types of molecular marker
. Non PCR Based
. PCR Based
RFLP
RAPD
AFLP
SSR
SNP
Conclusion
References
Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed, and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.
A genome is an organism’s complete set of DNA or complete genetic makeup, The entire DNA complement. It describes the identity and the sequence of genes of an organism.
Genomics is the study of entire genomes(structure, function, evolution, mapping, and editing of genomes)
Executing the sequencing and analysis of entire human genome enables more rapid and effective identification of disease associated genes and provide drug companies with pre validated targets.
Proteomics is the systematic high-throughput separation and characterization of proteins within biological systems./ large scale study of protein and their functions.
Proteomics measures protein expression directly, not via gene expression, thus achieving better accuracy. Current work uses 2-dimensional polyacrylamide gel electrophoresis(2D- PAGE) and mass spectrometry.
New separation and characterization technologies, such as protein microarray and high throughput chromatography are being developed.
description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it
Complete the table WHAT QUESTION DOES EACH ANSWER... WHAT IS THE G.pdfmurtuzadahadwala3
Complete the table
WHAT QUESTION DOES EACH ANSWER... WHAT IS THE GOAL FOR EACH...
Methods
Definition
Question that answers
Draw it
RNA seq
RNA seq or RNA sequencing is the examination of the RNA in a sample using next-generation
sequencing by analyzing the cellular transcriptome of RNA-encoded gene expression patterns.
MeDIP-Seq
MeDIP-Seq is an abbreviation that stands for Methylated DNA Immunoprecipitation
Sequencing. It is a technique used to examine patterns of DNA methylation in the genome.
Utilizing an antibody that binds specifically to methylated DNA, the DNA is then isolated and
sequenced. This enables researchers to identify methylated regions of the genome and investigate
their function. MeDIP-Seq can be used to answer queries such as how DNA methylation
influences gene expression, how it varies between cell types or tissues, and how it responds to
the environment.
HM450K
HM450K stands for HumanMethylation450 BeadChip by Illumina. It is a microarray-based
technique used to examine patterns of DNA methylation in the human genome. The device
contains probes that target over 450,000 CpG sites throughout the genome, enabling researchers
to identify methylated or unmethylated regions of the genome. HM450K can be used to answer
such queries as how DNA methylation patterns differ between healthy and diseased tissues, how
they change over time, and how they are influenced by the environment.
Mass Spec
The technique of mass spectrometry (MS) is used to analyze molecules based on their mass-to-
charge ratio. Utilized frequently in proteomics research to identify and quantify proteins in
biological samples. The method entails ionizing the sample molecules, separating them
according to their mass-to-charge ratio, and detecting them with a mass spectrometer. MS can be
used to determine which proteins are present in a biological sample, how much of each is
present, and how they interact.
EMSA
Electrophoretic Mobility Shift Assay stands for EMSA. It is a technique utilized to investigate
protein-DNA interactions. A labeled DNA probe is incubated with a protein extract from cells or
tissues, followed by electrophoresis on a polyacrylamide gel. If the protein binds to the DNA
probe, its electrophoretic mobility on the gel will change. EMSA can be used to determine which
proteins bind to a particular DNA sequence, as well as to investigate the kinetics and specificity
of protein-DNA interactions. This technique is frequently employed in the field of transcriptional
regulation, where it can be used to investigate how transcription factors bind to DNA and
regulate gene expression. Other protein-DNA interactions, such as those involved in DNA repair
or replication, can also be studied using EMSA. Overall, EMSA is an effective method for
investigating protein-DNA interactions and their function in cellular processes.
MNAse-Seq
MNase-Seq is an acronym that stands for Micrococcal Nuclease Sequencing. It is a technique
used to examine the structure and acce.
Molecular Markers and Their Application in Animal Breed.pptxTrilokMandal2
Molecular markers have had a significant impact on breed development and conservation efforts, transforming genetics and offering vital insights into genetic diversity, lineage tracing, and genotype characterization. The importance of molecular markers in improving genetic gains, facilitating breeding programs, and preserving genetic diversity for the long-term sustainability of the animal population has been underlined in this review paper. Emerging advancements in molecular marker technology show enormous potential for improving and conserving breeds. Deeper insights into the genetic basis of complex traits will be provided through GWAS, CRISPR/Cas9, gene editing technologies, and sequencing technologies, resulting in faster genetic gains. Breeders and conservationists will be able to make more informed judgments thanks to these technologies. In conclusion, molecular markers have had a significant impact on breed conservation and enhancement. Their innovations have changed the industry and given both conservationists and breeders vital knowledge. We can pave the road for more effective and sustainable genetic improvement and the preservation of biodiversity for future generations by combining the power of molecular markers with conventional breeding and conservation techniques.
Protein protein interaction, functional proteomicsKAUSHAL SAHU
IntroductionTypes of Protein-protein interactionsEffects of Protein-Protein InteractionsProtein-Protein Interaction Identification Methods :- Experimental (In vivo) Yeast two hybrid system- Experimental (In vitro) Co-immunoprecipitation, ChIP, Affinity Blotting, Protein Probing - Computational (In silico) Database of interacting proteins, VisANT etc.
ConclusionReferences
• It is a technique that predicts the interaction between a macromolecules and a chemical molecule.
• Most of the existing efforts to identify the binding sites in protein-protein interaction are based on analyzing the differences between interface residues and non-interface residues, often through the use of machine learning or statistical methods.
• Its major application is to Identify the protein ligand binding sites is an important process in drug discovery and structure based drug design.
• Earlier, detecting protein ligand binding site is expensive and time consuming by traditional experimental method. Hence, computational approches provide many effective strategies to deal with this issue.
Similar to Assays for protein dna interactions (20)
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
2. • Proteins interact with DNA at many points for critical
functions to maintain the overall integrity of the cell.
• This interaction may be specific or non specific.
• Some assays for determining protein-DNA interactions –
Chromatin
immunoprecipitation
Yeast one hybrid assay
Immunosorbant assay
DPI ELISA
In silico tools
SELEX
Protein binding assays
Xray crystallography
DAP-seq
DNA footprinting
7. Strengths and Limitations
• Allows observation of highly
dynamic events.
• Can be combined with
sequencing, PCR, cloning,
footprinting, microarray, etc.
• Only proteins for which antibodies
are available can be studied.
• Conformational changes on
binding may prevent antibody
binding.
9. • Construct 1/Bait plasmid: Bait+ Promoter + Reporter gene
BAIT REPORTER GENE
Bait is the
sequence of our
interest along
with promoter
Transformed
into yeast cells
Reporter gene will
be expressed
Pro
• Construct 2/Prey plasmid: Prey + AD + Marker
Prey is the
sequence encoding
for TF of interest
Yeast activating
domain [GAL4
usually]
Selection marker
Transformed into
pretransformed
yeast cells
10. BAIT REPORTER GENE
Pro
BAIT REPORTER GENE
Pro
Case 1: If the protein recognizes and binds
to SOI, transcription will result in
expression of reporter gene
Case 2: No transcription of reporter gene
will happen if protein can’t recognize SOI
11. Strengths and Limitations
• Can detect interactions between
non transcription proteins and
DNA.
• Compatible with many existing
libraries.
• Rates of false positive are high.
• Improper protein folding.
13. • Laboratory methods are very time consuming and very expensive.
• Low cost and efficient computational tools provide a way to study
protein-DNA complexes.
• There are many tools, software or web based, freely available for such
interactive studies.
• These tools uses structural information, model simulations,
thermodynamic parameters, experimental conditions, biochemical
properties, evolutionary info, scoring functions to predict protein-
DNA binding affinity quantitively.
14. • TRANSFAC (TRANScription FACtor database) – DB of eukaryotic TFS, their
binding sites, DNA binding profiles; helps in identifying potential TFBSs and in
transcriptional regulation.
• DISPLAR (DNA-Interaction Site Prediction from a List of Adjacent residues) –
Predicts the residues of a protein which interact with DNA.
• YEASTRACT (Yeast Search for Transcriptional Regulators And Consensus
Tracking) – DB of TFs and target genes of yeast, helps in determining
potential TFBSs.
• Some web based tools for DNA-binding protein interaction studies – DP-Bind,
HMMBinder, PiDNA, iDNA-Prot, PreDBA.
15. References
• Cozzolino, F., Iacobucci, I., Monaco, V., & Monti, M. (2021). Protein-DNA/RNA interactions: An
overview of investigation methods in the -omics era. Journal of Proteome Research, 20(6), 3018–
3030. https://doi.org/10.1021/acs.jproteome.1c00074
• Das, P. M., Ramachandran, K., vanWert, J., & Singal, R. (2004). Chromatin immunoprecipitation
assay. BioTechniques, 37(6), 961–969. https://doi.org/10.2144/04376RV01
• Dey, B., Thukral, S., Krishnan, S., Chakrobarty, M., Gupta, S., Manghani, C., & Rani, V. (2012). DNA-
protein interactions: methods for detection and analysis. Molecular and Cellular
Biochemistry, 365(1–2), 279–299. https://doi.org/10.1007/s11010-012-1269-z
• Emamjomeh, A., Choobineh, D., Hajieghrari, B., MahdiNezhad, N., & Khodavirdipour, A. (2019). DNA-
protein interaction: identification, prediction and data analysis. Molecular Biology Reports, 46(3),
3571–3596. https://doi.org/10.1007/s11033-019-04763-1
• Ferraz, R. A. C., Lopes, A. L. G., da Silva, J. A. F., Moreira, D. F. V., Ferreira, M. J. N., & de Almeida
Coimbra, S. V. (2021). DNA-protein interaction studies: a historical and comparative analysis. Plant
Methods, 17(1), 82. https://doi.org/10.1186/s13007-021-00780-z
• Pandey, P., Hasnain, S., & Ahmad, S. (2019). Protein-DNA Interactions. In Encyclopedia of
Bioinformatics and Computational Biology (pp. 142–154). Elsevier. https://doi.org/10.1016/b978-0-
12-809633-8.20217-3
• Watson, J. D., Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (2014). Molecular biology of
the gene (7th ed.). Pearson.
Good morning sir/ma’am, Myself Amaan Shaikh and my topic is assays for determining protein DNA interactions
Replication, transcription, translation, repair, regulation, etc….This interaction can be specific or non specific depending upon the functions..
As someone rightly said, life is a relation between molecules, not a property of any one molecule. So by learning proteins DNA interactions, humans will be a lil bit more closer to understand life, to understand different processes that happen within the cell.
And this understanding will help in designing novel/modified proteins with different specificity, affinities n functions for various purposes. These are some assays for determining protein DNA interactions. I am going to explain couple of em.
for example, DNA binding proteins in with different specificity, affinity, helping in transcriptional regulation, gene therapy functional genomics cellular functions including gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. The 3-dimensional structure of chromatin is maintained by the binding of histones and other regulatory proteins to the DNA. It is vital to know how DNA-binding proteins affect the functioning of any particular gene and to identify which particular protein binds to a specific DNA sequence in vivo
https://www.scripps.edu/barbas/pdf/Segal00COCB.pdf
R2
Chromatin immunoprecipitation (ChIP) has become a very widely used technique for determining the in vivo location of binding sites of various transcription factors. As we all know, chromatin is basically complex of DNA and protein complexes….in a lil bit, we will see what immunoprecipitation means here
So we have a DNA here, and some DBPs. They can bind and unbind to DNA but here, we require proteins DNA complexes. And thus, we use formaldehyde [or Methylene blue, acridine orange, UV, laser] as a cross linking agent to fix proteins on DNA after which cells are lysed, using ionic salts [Nacl] or buffering salts [tris HCL]…. ref5
R5- Next important step is fragmentation of DNA to 100-500bps fragments to pinpoint the location of the DNA sequence of interest. This can be done by sonication, RE digestion or by Nuclease
In sonication, sound energy (>20khz) is applied resulting in agitation, bubble formation, bubble growth, bubble break resulting in shearing forces
Here comes the immunoprecipitation part….we use Protein A/G coated beads specific to protein of interest to isolate and elute out protein-DNA complexes. Later unlinking of crosslinking agent [that is aldehyde] is done by incubating the protein-DNA complexes at higher temperature [68 degree Celsius accordingly] to get free DNA and protein. In post immunoprecipitation, proteins and DNA can be processed for sequencing in chipseq, chip Mass spec or chip micro array chips.
Then proteins are can be either digested or used in post immunoprecipitation analyses.
These are some strengths n limitations of this technique. It allows observation of minute-by-minute rapid changes at a single promoter. This technique can be combined with various other techniques to broaden the applications.
One of the disadvantage here is antibodies against protein of interest should be available. Also, as the protein binds to the DNA, some conformational change may hide the epitope and we may get false negative results.
Difficult to adapt for high throughput screening
This is powerful method to identify proteins [any proteins and not just transcription factors] that can interact with a DNA sequence of interest, is a modified version of yeast two hybrid system
which is used as a protein protein interaction assays
This system is kinda like fishing,,,, here we are providing a bait and prey may or may not take that bait. First construct consist of a bait which is a sequence of interest, promoter and a reporter gene..umm, let’s say…LacZ…this will help in detecting whether the bait is taken by prey or not. Bait construct is ligated with the integrative vector which is later transformed into a suitable yeasts cells.
2nd construct here is our prey [that is, protein of interest], yeast activating domain that brings polymerase, normally Gal4 promoter, and a selection marker for example HIS3. This 2nd construct is then transformed into yeast cells which were pretransformed with bait construct.
After culturing and selecting using a minimal medium without leucine, 2 cases are possible.
Case 1: So if the prey protein is capable of recognizing and binding to the bait, it will bind to it, activating domain will bring promoter and other transcription factors ultimately leading to the expression of reporter gene…lacZ in our case which can be assayed using the galactosidase assay.
Case 2: If prey can’t bind to the bait, there will be no expression of reporter gene so no signals in galactosidase assay.
Leu2 leucine, ura3 for uracil
C3, c4, cam pathway
So, by fusing protein(s) of interest to a strong activation domain allows Y1H to detect a variety of DNA-binding proteins, including those that do not directly function in transcription, e.g. replication proteins, DNA repair proteins, and repressor proteins.
Most Y1H experiments can use hybrid prey libraries [that is, prey with activating domains] that have been constructed for Y2H applications, e.g. Gal4p- or LexA-based protein libraries can also be used for screening against various DNA baits in Y1H.
Polymerase may bind to promoter leading to false positive results…and fusion with activating domain may result in conformation change so prey might not bind to the bait sequences, which might be capable of binding originally.
https://bitesizebio.com/25900/an-overview-of-the-yeast-one-hybrid-assay/
Instead of going for laboratory methods which are expensive and time consuming, we can opt for computational tools. These tools are low cost plus are an efficient way to study protein-DNA complexes in short time. There are many tools freely available for such purposes. All these tools uses structural information, biochemical and thermodynamical properties, model simulations, etc to score the sequence of interest using some scoring matrices and thus predicting where a particular protein is capable of binding on our sequence of interest.
https://www.nature.com/articles/s41598-020-57778-1
These are some of the tools that are available. TRANSFAC helps in identifying eukaryotic Transcription factor binding sites…similarly for yeasts, YEASTRACT, 3rd one.. is available…then there is Displar- which when provided with structure of protein known to bind DNA, can predict amino residues that t=interact with DNA with accuracy over 80%.....and lastly, some web based tools that help in protein-DNA interaction studies.
So, as the database and techniques increases and by using combinations of different techniques, along with modifications, protein DNA interactions can be studied efficiently.
r6
This are the references that I have used.
With this, I end my presentation. I hope I was clear with the basic principle at least. Thank you very much.
Myelin gene promoters – TFs, 10.1007/978-1-4939-9554-7_37
Whole TFs, then methylation dependent - https://pubmed.ncbi.nlm.nih.gov/14706630/
In silico DNA binding proteins generally