A presentation on the various interactions occurring during gene expression between proteins and DNA

1. PROTEIN – DNA
INTERACTIONS
DARIYUS KABRAJI
MSC-1 BATCH 2 ROLL NO 18

2. INTRODUCTION
• In the late 19th century, scientists microscopically observed
the association of proteins with DNA strands.
• Since then, researchers have used a variety of procedures
to demonstrate that proteins interact with DNA and RNA to
influence the structure and function of the corresponding
nucleic acid.
• Protein–nucleic acid interactions therefore play a crucial
role in central biological processes, ranging from the
mechanism of replication, transcription and recombination
to enzymatic events utilizing nucleic acids as substrates.

3. DNA BINDING PROTEIN
• DNA-binding proteins (DBPs), such as transcription factors,
constitute about 10% of the protein-coding genes in
eukaryotic genomes and play pivotal roles in the regulation
of chromatin structure and gene expression by binding to
short stretches of DNA.
• Sequencing of eukaryote genomes disclosed that about
10% of all genes encode potential DBPs. Hence, every
higher plant or vertebrate genome harbours over 2000 of
these DBP genes.
• Despite their importance in many fundamental processes,
e.g. during stress or disease, throughout development and
in controlling yield or growth, our knowledge on this
tremendous number of putative DBPs and their interaction
with DNA is limited

4. DNA BINDING PROTEIN

5. INTERACTION TYPES
SPECIFIC
• The sequence of
nucleotides directly affects
the interaction outcome
• Control transcription in
prokaryotes and
eukaryotes, mediated by
hydrogen bonding, ionic
interactions and Van der
Waal’s forces
NON SPECIFIC
• The sequence of nucleotides
does not matter, as far as the
binding interactions are
concerned
• Histone (protein) - DNA
interactions are an example of
such interactions, and they
occur between functional
groups on the protein and the
sugar-phosphate backbone of
DNA

6. SPECIFIC INTERACTION
• In humans, Replication protein A is the best-understood
member of this family and is used in
processes where the double helix is separated
• The DNA binding proteins in these processes
include transcription factors
• These binding proteins seem to stabilize single-stranded
DNA and protect it from forming stem-loops
or being degraded by nucleases
• This process is used in DNA replication,
Transcription and repair

7. SPECIFIC INTERACTION

8. LEVELS OF SPECIFICITY
The factors considered to assess the levels of
specificity in a DNA- protein interaction are:
• Site specification
• Recognition
• Affinity
• Equilibrium selection

9. NON SPECIFIC INTERACTIONS
• Within chromosomes, DNA is held in complexes with structural
proteins. These proteins organize it into chromatin. In eukaryotes this
structure involves DNA binding to a complex of small basic proteins
(histones), while in prokaryotes multiple types of proteins are involved.
• The histones form a disk-shaped complex nucleosome, which contains
two turns of ds DNA wrapped around its surface. These non-specific
interactions are formed through basic residues in the histones making
ionic bonds to the acidic sugar-phosphate backbone of the DNA, and
are independent of the base sequence.
• Chemical modifications of these basic amino acid residues include
methylation, phosphorylation and acetylation. These chemical changes
alter the interaction strength & make the DNA more or less accessible
to transcription factors and changing the rate of transcription

10. NON SPECIFIC INTERACTIONS

11. CHROMATIN REMODELLING
• It s the modification of chromatin architecture to allow
access of condensed genomic DNA to the regulatory
transcription machinery proteins, and thus control gene
expression
• Two types: Covalent Histone Modifying Complexes & ATP
dependant remodelling complexes
• Histone Acetyl Transferases (HAT) bring about covalent
histone modifications by binding themselves to the DNA
• This is an important interaction since dynamic
remodelling of chromatin imparts an epigenetic
regulatory role in several key biological processes, and
irregularities can result in inconveniences

12. PROCEDURE
• Specific histone-modifying complexes catalyse the
addition or deletion of various chemical elements on
histones.
• These enzymatic modifications include acetylation,
methylation, phosphorylation, and ubiquitination and
occur at the nucleosome
• Such modifications affect the binding affinity between
histones and DNA, and thus loosening and tightening
the condensed DNA wrapped around histones
• Observed by mass spectrometry (measures the mass-to-
charge ratio and abundance of gas-phase ions)

13. PROCEDURE

14. DETECTION METHODS
1. Chromatin Immunoprecipitation (ChIP)Assays:
• The ChIP method can be used to monitor transcriptional
regulation through histone modification.
• The ChIP assay method allows analysis of DNA-Protein
interactions in living cells by treating the cells with
formaldehyde to stabilize the interactions for detection.
• It requires knowledge of the target protein and DNA
sequence which will be analysed, to provide an antibody
against the protein of interest to selectively precipitate the
protein-DNA complex from the other genomic DNA
fragments and protein-DNA complexes, which can be
amplified by PCR.

15. DETECTION METHODS
2. DNA Electrophoretic Mobility Shift Assay (EMSA):
• The EMSA studies proteins binding to known DNA
oligonucleotide probes and assesses the specificity of the
interaction.
• The technique is based on the principle that protein-DNA
complexes migrate more slowly than free DNA molecules when
subjected to polyacrylamide or agarose gel electrophoresis.
Because the rate of DNA migration is retarded upon protein
binding, the assay is also called a gel retardation assay.
• Adding a protein-specific antibody to the binding components
creates an even larger complex (antibody-protein-DNA) which
migrates even slower during electrophoresis, this is known as a
supershift and can be used to confirm protein identities.

16. DETECTION METHODS
3. DNA Pull-down Assay:
• Pull-down assays use a DNA probe labelled with a high affinity tag,
such as biotin, which allows the probe to be recovered or
immobilized. A DNA probe can be complexed with a protein from a
cell lysate in a reaction similar to that used in the EMSA and then
used to purify the complex using agarose or magnetic beads.
• The proteins are then eluted from the DNA and detected by
Western blot or identified by mass spectrometry.
• Alternatively, the protein may be labelled with an affinity tag or the
DNA-protein complex may be isolated using an antibody against the
protein of interest (similar to a supershift assay). In this case, the
unknown DNA sequence bound by the protein is detected by
Southern blotting or through PCR analysis.

17. DETECTION METHODS
4. Reporter Assay:
• Reporter assays provide a real-time in vivo read-out of
translational activity for a promoter of interest.
• Reporter genes are fusions of a target promoter DNA
sequence and a reporter gene DNA sequence which is
customized by the researcher and the DNA sequence codes
for a protein with detectable properties like firefly /Renilla
luciferase or alkaline phosphatase. These genes produce
enzymes only when the promoter of interest is activated.
• The enzyme, in turn, catalyses a substrate to produce either
light or a colour change that can be detected by spectroscopic
instrumentation. The signal from the reporter gene is used as
an indirect determinant for the translation of endogenous
proteins driven from the same promoter.

18. DETECTION METHODS
5. Microplate Capture and Detection Assays:
• A mix of the pull down assay and ELISA, microplate capture assays use
immobilized DNA probes to capture specific protein-DNA interactions
and confirm protein identities and relative amounts with target specific
antibodies.
• Typically, a DNA probe is immobilized on the surface of 96- or 384-well
microplates coated with streptavidin. A cellular extract is prepared and
added to allow the binding protein to bind to the oligonucleotide. The
extract is then removed and each well is washed several times to
remove non-specifically bound proteins.
• Finally, the protein is detected using a specific antibody labelled for
detection. This method can be extremely sensitive , detecting less than
0.2pg of the target protein per well. This method may also be utilized
for oligonucleotides labelled with other tags, such as primary amines
that can be immobilized on microplates coated with an amine-reactive
surface chemistry.

19. DETECTION METHODS
6. DNA Footprinting:
• Footprinting is one of the most widely used methods for obtaining
detailed information on the individual nucleotides in protein–DNA
complexes, even inside living cells. In such an experiment,
chemicals or enzymes are used to modify or digest the DNA
molecules.
• When sequence specific proteins bind to DNA they can protect the
binding sites from modification or digestion. This can subsequently
be visualized by denaturing gel electrophoresis, where unprotected
DNA is cleaved more or less at random.
• Therefore it appears as a ‘ladder’ of bands and the sites protected
by proteins have no corresponding bands and look like foot prints in
the pattern of bands. The foot prints there by identify specific
nucleosides at the protein–DNA binding sites

20. CONCLUSION
• The entire hierarchy of specificity criteria must be
considered during examination of DNA - protein
interactions.
• There are several methods used to specifically detect
the DNA binding proteins (DPB) and isolate them from
the sample. Identifying these proteins and their
manner of interaction with the nucleic acid sheds light
on the genes expressed and the functions that arise
from the expression
• However, each method has their own limitations and
new extraction methods are being tested for more
sensitive and accurate results

21. FUTURE PROSPECTS
Confirming the Functional Importance of a Protein–DNA
Interaction:
• Correlation between nucleotides required for protein
binding and those required for activity of the control
element
• Trans-activation of a reporter gene or endogenous
gene by over-expression of a DNA-binding protein
• Cooperative binding and synergistic function of
proteins bound to adjacent control elements
• Comparison of genomic and in vitro Footprinting
patterns