EMBRYOLOGY

HNS 2126
ANNASTACIA MBISI
Molecular regulation & signaling

• Learning objectives
Review of composition & structure of nucleic acid
Gene transcription
Regulators of gene expression
Embryonic induction
Composition and Structure of
nucleic acid
• Nucleic acid is huge organic molecules that contain
carbon, hydrogen, oxygen, nitrogen, and
phosphorus.
• It is a chain of repeating monomers called nucleotides
• There are two types of Nucleic Acids:
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
Nucleotides

• Each nucleotides consists of three

components
Nitrogen base
Pentose sugar
Phosphate group
Nitrogenous base

• Nucleic acid contains four different

nitrogenous bases, which contain atoms
of C, H, O, and N.
• The bases are divided into two categories
Purines - larger, double –ring
Pyrimidines - smaller single ring
DNA bases

• Purines-adenine(A) and guanine(G)-double

carbon nitrogen genes
• Pyrimidines -Thymine (T) and cytosine(C))-
single carbon nitrogen rings
• There are about 3 billion DNA bases in a
human genome
RNA bases

• Purines-adenine(A) and guanine(G)

• Pyrimidines- Uracil (U) and cytosine(C))
Pentose sugar

• A five-carbon sugar
• It attaches to each base in the nucleic acid
• In the DNA the pentose sugar is called
deoxyribose
• In the RNA it is called ribose
Phosphate groups

• Phosphate groups (PO4) alternate with

pentose sugars to form the “backbone”
of a DNA /RNA strand;
• The bases project inward from the
backbone chain (pentose sugar)
Deoxyribonucleic acid(DNA)
• A double helix nucleic acid located in the
nucleus and mitochondria
• Carries genetic information that directs
manufacture of proteins
• Each 3 bases in a row specify the code for a
particular amino acid
Structure of DNA (watson- crick
model)
• Double helix model
• DNA can be envisioned as a twisted ladder
with chemical bonds as its rungs.
• Sugar and phosphate molecules held by a
strong phosphodiester bonds make the
two sides of the ladder
Structure of DNA cont’d

• Nitrogenous bases project from each side of

the ladder at regular intervals
• Nitrogenous bases held together by weak
hydrogen bonds form the rungs
Adenosine=Thymine-2hydrogen bonds
Guanine=Cytosine-3 hydrogen bonds
DNA as the genetic code

• DNA direct the synthesis of all the body’s

proteins
• Proteins contain polypeptides which are in
turn composed of sequences of amino acids
The body contain 20 amino acids the
sequence of which is determined by DNA
DNA as the genetic code
cont’d
Formation of an amino acid is directed by
a triplets of bases(codons)
There are 64 codons three of which signal
the end of a gene: Termination or
nonsense codons
DNA code is universal- all living organisms
use the same codes to specify proteins
DNA as the genetic code
cont’d
• Mitochondria have their own
extranuclear DNA that codes for AA that
differ from those of the same nuclear DNA
codons
DNA replication

• Occurs during cell division

• It involves breaking of the weak hydrogen
bonds leaving a single strand.
• This is followed by complementary base
pairing.
DNA replication cont’d

• DNA polymerase-travels along the DNA

strand:
• Adding correct nucleotides to the free
end of the new strand
• Proofreading –ensure that the base is
actually complementary to the template
base
From genes to proteins

• Protein synthesis involves two process

Transcription
Translation
Transcription

• Process by which mRNA is synthesized from a

DNA template
• RNA polymerase bind to a promoter site on the
DNA-specifies the beginning of a gene; then pulls
the portion of DNA strand apart from one another
thus exposing the unattached DNA bases
Transcription cont’d

• This continues until a termination

sequences is reached;
RNA polymerase then detaches from the
DNA and the transcribed mRNA freed to
move out of the nucleus into the
cytoplasm
Gene splicing

• Before the mRNA leaves the nucleus many

of the RNA sequences are removed by
nuclear enzymes
• The remaining sequences are spliced
together to form the functional mRNA that
migrates to the cytoplasm
Gene splicing cont’d

• Introns-excised sequence; do not code for

proteins
• Exons-sequences that are left; code for
proteins.
Translation
• Process by which RNA directs the synthesis of a polypeptide.
• mRNA cannot directly code for AA hence it interacts with tRNA
• tRNA molecule has a site for the attachment of an AA; at the
opposite side is a sequence of 3 nucleotides(anti-codon)
• Anti-codon undergoes complementary base pairing with an
appropriate codon in mRNA
Translation cont’d

• Actual site of protein synthesis is the ribosome

• The ribosome first binds to an initiation site
on mRNA sequence
• Then bind the tRNA to its surface so that base
pairing between the two can occur
Translation cont’d

• The ribosome then moves along the mRNA

sequence codon by codon and as each
codon is processed an AA is translated.
• The ribosome then provides covalent
peptide bonds between adjacent AA
resulting in a growing polypeptide
Translation cont’d
• When ribosome arrives at a termination signal on the
mRNA sequence translation and polypeptide formation
cease
• mRNA, ribosome and polypeptide separate from one
another.
• Polypeptide is released into the cytoplasm to perform
its required function
Regulation of gene
expression
Introduction

• Gene expression is the process of

“turning on” a gene to produce mRNA and
protein
• Each somatic cell in the body generally
contains the same DNA
Introduction cont’d

• Although each cell in the body contains the

same DNA sequences, each cell does not
turn on, or express, the same set of genes
• In other words, in any given cell, not all genes
encoded in the DNA are transcribed into
mRNA or translated into protein
Overview of regulation of gene
expression
• For a cell to function properly, necessary
proteins must be synthesized at the proper
time
• All cells control or regulate the synthesis of
proteins from information encoded in their
DNA
Overview cont’d

• Whether in a simple unicellular organism or

a complex multi-cellular organism, each
cell controls when its genes are expressed,
how much of the protein is made, and
when it is time to stop making that
protein because it is no longer needed.
Overview cont’d

• The regulation of gene expression conserves energy and space

It is more energy efficient to turn on the genes only when
they are required
Additionally, only expressing a subset of genes in each
cell saves space because DNA must be unwound from its
tightly coiled structure to transcribe and translate the
DNA
Regulation of gene expression

• It can happen at any of the stages as DNA is

transcribed into mRNA and mRNA is translated into
protein.
• For convenience, regulation is divided into five levels:
1. Epigenetic
2. Transcriptional
Regulation of gene
expression cont’d
3. Post-transcriptional
4. Translational
5. Post-translational
1. Epigenetic(“Around genetics”)
control
• It involves changes to genes that do not
alter the nucleotide sequence of the
DNA and are not permanent.
• Instead, these changes alter the
chromosomal structure so that genes can
be turned on or off
Level of Epigenetic control
• Occurs through heritable chemical modifications of
the DNA and/or chromosomal proteins
• DNA modification-Example: methylation
The addition of methyl groups to the DNA
Interestingly, methylation patterns can be passed on
as cells divide
Chemical modification of DNA cont’d

Other heritable chemical modifications of

DNA may also occur.
Modification of chromosomal
protein
• Example: Modification of Histone
Proteins
• Histones are chromosomal proteins that
tightly wind DNA so that it fits into the
nucleus of a cell
Modification of Histone Proteins

• The human genome, for example, consists

of over three billion nucleotide pairs.
• An average chromosome contains 130
million nucleotide pairs, and each body cell
contains 46 chromosomes.
Modification of Histone
Proteins cont’d
• If stretched out linearly, an average human
chromosome would be over four
centimeters long.
• In order to fit all of this DNA into the nucleus
of a microscopic cell,
the DNA must be tightly wound around
proteins.
Modification of Histone
Proteins cont’d
It is also organized so that specific segments
can be accessed as needed by a specific cell
type(fig.1)
Fig 1
Mod. of histone proteins
cont’d
• The first level of organization, or packing, is
the winding of DNA strands around
histone proteins.
• Histones package and order DNA into
structural units called nucleosome
complexes, which can control the access of
proteins to the DNA regions [Fig. 2(a)]
Mod. of histone proteins
cont’d
• Under the electron microscope, this winding of DNA
around histone proteins to form nucleosomes
looks like small beads on a string [Fig 2(b)].
• These beads (histone proteins) can move along
the string (DNA) and change the structure of the
molecule
Fig.2
Mod. of histone proteins
cont’d
• If a gene is to be transcribed, the
nucleosomes surrounding that region of DNA
can slide down the DNA to open that specific
chromosomal region
• This allows access for RNA polymerase
and other proteins, (transcription
factors), to bind to the promoter region
and initiate transcription
Mod. of histone proteins
cont’d
• If a gene is to remain turned off, or silenced,
the histone proteins and DNA have
different modifications that signal a closed
chromosomal configuration.
• In this closed configuration, the RNA
polymerase and transcription factors do
not have access to the DNA and
transcription cannot occur(fig.3)
Fig. 3
Mod. of histone proteins
cont’d
• How the histone proteins move is dependent on
signals found on the histone proteins.
• These signals are “tags” – in the form of
phosphate, methyl, or acetyl groups – that
open or close a chromosomal region (Fig 3).
Mod. of histone proteins
cont’d
• These tags are not permanent, but may be
added or removed as needed.
• Since DNA negatively charged, changes
in the charge of the histone will change how
tightly wound the DNA molecule will be.
Mod. of histone proteins
cont’d
• When unmodified, the histone proteins
have a large positive charge; by adding
chemical modifications like acetyl groups,
the charge becomes less positive.
2. Transcriptional control

• It is control of whether or not an mRNA is

transcribed from a gene in a particular cell
• The transcription of genes requires an RNA
polymerase to bind to a promoter to
initiate transcription
Transcriptional control cont’d

• RNA polymerase requires other proteins,

or transcription factors, to facilitate transcription
initiation
Transcription factors are proteins that bind to
the promoter sequence and other regulatory
sequences to control the transcription of the
target gene
Transcriptional control cont’d

Transcription factors must bind to the

promoter region first and recruit RNA
polymerase to the site for transcription
to begin
The promoter and transcription factors

• The promoter region is immediately

upstream of the coding sequence and ranges
from a few to hundreds of nucleotides long.
• The length of the promoter is gene-specific
and can differ dramatically between genes
The promoter and
transcription factors cont’d
• The longer the promoter, the more
available space for proteins to bind
• Consequently, the level of control of gene
expression can differ quite dramatically
between genes.
The promoter and transcription
factors cont’d
• The purpose of the promoter is to bind
transcription factors that control the
initiation of transcription (fig.4 top)
Top. Each gene
has a promotor
upstream of the
coding sequence.
The promoter
binds to
transcription
factors and helps
RNA polymerase
to bind and start
transcription
Bottom. Many
genes also have
upstream
enhancers.
Enhancers bind
activators, bend
around, and help
RNA polymerase
start
transcription.
The promoter and transcription
factors cont’d
• Within the promoter region, just upstream of the
transcriptional start site, resides the TATA box.
• This box is simply a repeat of thymine and
adenine dinucleotides (literally, TATA repeats).
• Transcription factors bind to the TATA box,
assembling an initiation complex
The promoter and transcription
factors cont’d
• Once this complex is assembled, RNA polymerase
binds to its upstream sequence and becomes
phosphorylated.
• This releases part of the protein from the DNA,
activates the transcription initiation complex, and
places RNA polymerase in the correct orientation to
begin transcription (Fig.4, top)
Enhancers and repressors

• Enhancers-these are regions that help

increase transcription and are not necessarily
close to the genes; they can be located
thousands of nucleotides away
• They can be found upstream, within the
coding region, or downstream of a gene
Enhancers and repressors
cont’d
• Enhancers are binding sites for activators
• When an enhancer is far away from a gene, the
DNA folds such that the enhancer is brought into
proximity with the promoter, allowing interaction
between the activators and the transcription
initiation complex (Fig 4 bottom).
Enhancers and repressors
cont’d
• Transcriptional repressors can bind to
promoter or enhancer regions and block
transcription.
• Both activators and repressors respond to
external stimuli to determine which genes
need to be expressed
3. Post-transcriptional
control
• Occurs after the mRNA is transcribed but
before translation begins.
• This regulation can occur at the level of
mRNA processing, transport from the
nucleus to the cytoplasm, or binding to
ribosomes.
Mechanism of Post-transcriptional
control
a) Alternative RNA splicing
b) Control of RNA Stability
a) Alternative RNA splicing
• It is a mechanism that allows different
combinations of introns, and sometimes
exons, to be removed from the primary
transcript (Fig. 5).
• This allows different protein products to be
produced from one gene.
Figure 5
Alternative RNA splicing
cont’d
• Alternative splicing can act as a mechanism
of gene regulation.
• Differential splicing is used to produce
different protein products in different cells or
at different times within the same cell
Alternative RNA splicing
cont’d
• Alternative splicing is now understood to be
a common mechanism of gene regulation in
eukaryotes; up to 70 percent of genes in
humans are expressed as multiple proteins
through alternative splicing.
Control of RNA stability in the
cytoplasm
• The longer an mRNA exists in the
cytoplasm, the more time it has to be
translated, and the more protein is made.
• Many factors contribute to mRNA stability,
including the length of its poly-A tail.
Figure 5. The protein-coding
region of mRNA is flanked by 5′ and
3′ untranslated regions (UTRs).
RNA-binding proteins at the 5′ or
3′ UTR influence the stability of the
RNA molecule.
Control of RNA stability cont’d

• Proteins, called RNA-binding proteins (RBPs)

can bind to the regions of the RNA just upstream
or downstream of the protein-coding region.
• These regions in the RNA that are not translated
into protein are called the untranslated
regions, or UTRs
Control of RNA stability cont’d

• The region just before the protein-coding

region is called the 5′ UTR, whereas the
region after the coding region is called the 3′
UTR (Figure 5).
Control of RNA stability cont’d

• The binding of RBPs to these regions can

increase or decrease the stability of an
RNA molecule, depending on the specific
RBP that binds.
Control of RNA stability cont’d

• microRNAs, or miRNAs, can also bind to the

RNA molecule.
miRNAs are short (21–24 nucleotides) RNA
molecules that are made in the nucleus as
longer pre-miRNAs and then chopped into
mature miRNAs by a protein called dicer
Control of RNA stability cont’d

• miRNAs bind to mRNA along with a

ribonucleoprotein complex called the RNA-
induced silencing complex (RISC).
• The RISC-miRNA complex rapidly degrades
the target mRNA
4. Translational control

• After an mRNA has been transported to the

cytoplasm, it is translated into proteins.
• Control of this process is largely dependent on the
mRNA molecule.
• As previously discussed, the stability of the mRNA will
have a large impact on its translation into a protein
4. Translational control cont’d

• Translation can also be regulated at the level

of binding of the mRNA to the
ribosome.
• Once the mRNA bound to the ribosome, the
speed and level of translation can still be
controlled.
Translational control cont’d

• An example of translational control occurs in

proteins that are destined to end up in an
organelle called the endoplasmic
reticulum (ER).
• The first few amino acids of these proteins
are a tag called a signal sequence.
Translational control cont’d

• As soon as these amino acids are translated, a

signal recognition particle (SRP) binds to the
signal sequence and stops translation while the
mRNA-ribosome complex is shuttled to the ER.
• Once they arrive, the SRP is removed and translation
resumes
5. Post-translational control

• This type of control involves modifying the

protein after it is made, in such as way as to
affect its activity
• One example of post-translational regulation
is enzyme inhibition
5. Post-translational control cont’d

• When an enzyme is no longer needed, it is

inhibited by a competitive or allosteric
inhibitor, which prevents it from binding to its
substrate.
• The inhibition is reversible, so that the enzyme
can be reactivated later.
5. Post-translational control cont’d

• This is more efficient than degrading the

enzyme when it is not needed and then making
more when it is needed again
• The activity and/or stability of proteins can also
be regulated by adding functional groups,
such as methyl, phosphate, or acetyl groups
5. Post-translational control cont’d

• Sometimes these modifications can regulate

where a protein is found in the cell—for
example, in the nucleus, the cytoplasm, or
attached to the plasma membrane.
5. Post-translational control
cont’d
• The addition of an ubiquitin group to a
protein marks that protein for degradation.
• Ubiquitin acts like a flag indicating that the
protein’s lifespan is complete
5. Post-translational control cont’d

• Tagged proteins are moved to a

proteasome, an organelle that degrades
proteins (Fig. 6).
• One way to control gene expression,
therefore, is to alter the longevity of the
protein.
Fig. 6 proteins with ubiquitin tags are marked for degradation within the
proteasome.
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