GENE EXPRESSION AND

PROTEIN SYNTHESIS

Sumbong, Catherine Kate

Rada, Ericca Cialette Lara
Melocoton, Sky Marie
Flores, John Bernard
Benoman, Denzell Joevert
HOW DOES DNA LEAD TO RNA
AND PROTEIN?
The Central Dogma of Molecular Biology
 the dogma states that the information
contained in DNA molecules is transferred
to RNA molecules and then from the RNA
molecules the information is expressed in
the structure of proteins.
Gene Expression
 activation of a gene
 process by which the instructions in our
DNA are converted into a functional
product, such as a protein.
Two Key Steps involved in making Proteins
 Transcription
 Translation
A. Transcription
 transcribing the information from the
DNA molecule into a molecule of
messenger RNA
 RNA is a chemical similar in structure and
proteins to DNA, but it only has a single
strand and instead of the base thymine
(T), RNA has a base called uracil (U).
B. Translation
 The process in which information
encoded in an mRNA molecule is used to
assemble a specific protein.
 The mRNA serves as a template on which
the amino acid are assembled in proper
sequence and read by another type of
RNA, transfer RNA.
HOW IS DNA TRANSCRIBE INTO
RNA?
 Transcription starts when DNA double helix
begins to unwind
 Specific binding proteins bind to the
nucleosomes, making the DNA less dense
and more accessible. Only then the enzyme,
helicase unwinds the double helix.
Template Strand & anti sense strand
 it is the strand that serves as a template
for the formation of RNA
Coding strand & sense strand
 a strand with a sequence that matches
the RNA that will be produced
Ribonucleotides assemble along the
unwound DNA strand in the complementary
sequence
 C-G
 A-U
 T-A
IN EUKARYOTES
RNA Polymerase I (Pol 1)
 Catalyzes the formation of most of RNA
RNA Polymerase II (Pol II)
 Catalyzes mRNA formation
RNA Polymerase III (Pol III)
 Catalyzes tRNA and snRNA
Eukaryotic gene has two major parts
Structural gene
 which is transcribed into RNA
 it is made of exons and introns
Regulatory
 controls the transcription
Control Elements
Promoter (Initiation)
 The purpose of the promoter is to bind
transcription factors that control the initiation
of transcription.
 On the DNA strand, there is always a sequence
of bases that polymerase recognizes as an
initiation signal saying, “Start here”.
 They contain consensus sequences such as
TATA box which gets its name from the
sequence beginning TATAAT.
 Three RNA Polymerases interact with their
promoter regions via transcription factors.
Enhancer (Initiation)
 A DNA sequence that can be far removed
from the promoter region
 Also binds to transcription factors
enhancing transcription above the basal
level
 Pol 2 enzyme is still in its
unphosphorylated form
Elongation
 Once RNA polymerase is in position at the
promoter, the next step of transcription—
elongation—can begin. Basically,
elongation is the stage when the RNA
strand gets longer .
 The RNA transcript is nearly identical to
the non-template, or coding, strand of
DNA. However, RNA strands have the
base uracil (U) in place of thymine (T), as
well as a slightly different sugar in the
nucleotide. So, as we can see in the
diagram above, each T of the coding
strand is replaced with a U in the RNA
transcript.
 Phosphorylation of Pol 2
Transcription Termination
 so at the end of the gene is a terminal
sequence that tells the enzyme to “stop the
transcription”
 RNA polymerase will keep transcribing until
it gets signals to stop. The process of ending
transcription is called termination, and it
happens once the polymerase transcribes a
sequence of DNA known as a terminator.
 Pol 2 is dephosphorylated by a phosphatase
 Post-transcriptional Process
 The RNA transcription products are not
necessarily the functional RNAs. To
ensure that mRNA is functional, a 5'
cap is added to the beginning of the RNA
transcript, and a 3' poly-A tail is added to
the end.
 Once the two ends are capped, some
sections of the RNA transcript (introns)
are removed, and the remaining sections
(exons) are stuck back together.
What is the Role of RNA
Translation?
 Translation
 the process by which the genetic
information preserved in the DNA and
transcribed into mRNA is converted to the
language of proteins— that is the amino
acid sequence.
 tRNA, rRNA & mRNA participate in the
process.
 Ribosomes
 The synthesis of protein takes place in the
ribosomes. These spheres dissociate into
two parts– a larger (60s ribosome) and
smaller (40s ribosomes) body which
contains rRNA and polypeptide chains that
act as enzymes speeding up the synthesis.
 Triplets of bases on the mRNA are called
codons.
 provides a set of handy slots where tRNAs
can find their matching codons on the mRNA
template and deliver their amino acids.
 Transfer RNA (tRNA)
 connect mRNA codons to the amino acids
they encode.
 One end of each tRNA has a sequence of
three nucleotides called an anticodon, which
can bind to specific mRNA codons.
 Aminoacyl-tRNA synthetases recognize
specific tRNA molecules and amino acids to
be attached to the terminal group of tRNA
forming an ester bond
 Codon recognition site is a sequence of
three bases called an anticodon located at
the opposite end of the molecule in the
three dimensional structure of RNA.
What is the Genetic Code?

 Genetic Code is the sequence of

triplets of nucleotides (codons) that
determines the sequence of amino acids
in a protein.
 Codon - each amino acid is coded for by
a sequence of three bases.
 Genetic code is almost universal. The
universality of the genetic code implies
that all living matter on Earth arose from
the same primordial organisms.
 There are 20 amino acids in proteins, but
64 possible combinations of four bases
into triplets. All 64 codons (triplets) have
been deciphered.
 Three of them --- UAA, UAG and UGA ---
are “stop signs”. They terminate protein
synthesis.
 The initiation sign is AUG, which is also the
codon for the amino acid methionine. This
means that in all protein synthesis, the first
amino acid initially put into the protein is
always methionine. Methionine can also be put
in the middle of the chain.
How is Protein Synthesized?

ACTIVATION
 Each amino acid is activated by reacting
with ATP
 tRNA synthetase enzyme attaches
activated amino acid to own particular
RNA.
 INITIATION
 Forming the pre initiation complex
 To initiate the protein synthesis, a unique tRNA is used,
tRNA met I (I stands for initiator) is used for beginning protein
synthesis.

 Migration to mRNA
 The pre initiation complex binds to the mRNA. The ribosomes
is aligned on the mRNA by recognizing a special RNA sequence
called Shine-Dalgarno sequence which is complementary to a
sequence on the 30S ribosomal submit.

 Forming the full ribosomal complex

 The 50S ribosomal body joins the 30S ribosomal complex. The
complete ribosome carries three sites. The P-site is where the
growing peptide chain will bind.
 The A (acceptor) site, accepts the incoming tRNA bringing
the next amino acid.
 ELONGATION
 Binding to the A-site
 The A-site is vacant and each of the aminoacyl-tRNA
molecules can try to fit itself in.
 Forming the first peptide bond
 At the A-site, the new amino acid is bonded to the
fMet in a peptide bond by the enzyme peptidyl
transferase. The empty tRNA remains on the P site.
 Translocation
 The whole ribosome moves one codon along the
mRNA. The dipeptide is translocated from the A-site
to the P-site. The empty tRNA is moved to the E-site.
 Forming the second peptide bond
After the translocation, the A-site is associated with
the next codon on the mRNA.
 TERMINATION

After the final translocation, protein

synthesis terminates once a stop codon has
been encountered. (UAA, UGA or UAG) No
more amino acids can be added. The tRNA
itself is released from the P-site. At the end,
the whole mRNA is released from the
ribosome.
HOW ARE GENES REGULATED?
 Every embryo that is formed by sexual
reproduction inherits its genes from both the
parent sperm and the egg cells.
 The genes in its chromosomal DNA are not
active at all time.
 They are switched on and off during the
development and growth of the organism.
 After formation of the embryo, the cells
begin to differentiate. Some cells will
become neurons, some become muscle
cells, some become liver cells, and so on.
 Each cell must switch some of its gees on
and of either permanently or temporarily.
How this is done is the subject of gene
regulation.
 GENE REGULATION- the control process by
which the expression of a gene is turned on or
off.
 Many gene regulations occur at the
transcriptional level. (DNA RNA)
 Others operate at the translational level
(Mrna protein)
A. Control at the transcriptional level
 In eukaryotes, transcription is regulated by
three entities: PROMOTER, ENHANCER,
and RESPONSE ELEMENTS.

1. Promoters of a gene are located adjacent

to the transcription site. They are defined
by an initiator and conserved sequence. In
eukaryotes, the enzyme RNA polymerase
has little affinity for binding to DNA.
 GENERAL TRANSCRIPTION
FACTOR (GTF)
- these proteins form a complex with
RNA polymerase and the DNA and help
to position the RNA polymerase
correctly and stimulate the initiation of
transcription.
 Six transcription factor must bind to the
DNA and RNA polymerase to initiate
transcription.
 They first form the pre-initiation
complex. The critical event in starting the
transcription is the conversion to the
open complex.
2. ENHANCER- another group of
transcription factors speed up the
transcription process by binding the DNA
sequences that may be located several
thousand nucleotides away from the
transcription site.
 SILENCER- other DNA sequences bind
transcription factors but have the
opposite effect, they slow down
transcription.
3. RESPONSE ELEMENT- these enhancers
are activated by their transcription factors in
response to an outside stimulus.
4. Transcription does not occur at the same
rate throughout the cell’s entire life cycle.
Instead it is accelerated or slowed down at
the need arises.
 The interaction between the protein and
the DNA involves nonspecific electrostatic
interaction as well as more specific
hydrogen bonding.
 The transcription factors find their target
sites by twisting their protein chains so that
a certain amino acid sequence is present at
the surface. One conformational twist is
provided by metal-binding fingers.
 METAL-BINDING FINGGERS- these finger shapes
are created by ions, which form covalent bonds
with the amino acid side chains of the proteins.
 the zinc fingers interact with specific DNA
sequences. Zinc fingers allow the proteins
to bind in the major groove of DNA.
 Aside from metal-binding fingers, at least
two other prominent transcription factors
exist: helix-turn-helix and leucine zipper.
B. CONTROL ON THE POST-TRANSCRIPTIONAL
LEVEL
 The odds-on favorite would have been 100,000 to
150,000 genes.
 It was known that human produce 90,000
different proteins.
 The dogma stated that “one gene leads to one
mRNAleads to one protein” the only exception to
this rule were thought to occur in the production
of antibodies and other immunoglobulin based
proteins. These proteins were known to undergo
a type of post-transcriptional modification called
alternative splicing.
 ALERNATIVE SPICING provides another powerful
technique for controlling gene regulation.
C. Control on the Translational Level
1. The Specificity of a tRNA for its unique
amino acid
 First, achieved attachment of the proper
amino acid to the proper tRNA
 Aminoacyl-tRNA synthetase (AARS)
 Enzyme that catalyzes this reaction
 Recognize their tRNAs by specific nucleotide
sequences
 Active site of the enzyme
 Two sieving portions
Example:
 Isoleucyl-tRNA synthetases
 First sieve excludes any amino acids that are
larger than isoleucine
 If similar amino acid, valine which is smaller
than isoleucine, arrives at the active site, the
second sieve eliminates it.
 Second sieving is thus a “proofreading site”
2. Recognition of the stop codon
 Recognized by release factors otherwise a
longer polypeptide chain could be toxic.
 GTP and peptidyl-tRNA ester bond are
hydrolyzed
 This hydrolysis releases the polypeptide
chain and deacylated-tRNA
 The ribosome dissociates from the mRNA.
 Stop codon is used to continue translating
by inserting a very uncommon amino acid,
such as selenocysteine.
3. Post-translational Controls
a) Removal of methionine
 Methionine aminopeptidase
 Special enzyme that cleaves the peptide bond

b) Chaperoning
 Chaperones
 Help the newly synthesized polypeptide chains
to fold properly.
 Recognize hydrophobic regions
c) Degradation of misfolded proteins
 Proteasomes
Include a number of protein subunits
with proteolytic activity in the core of the
cylinder
Chaperones fails, proteases may degrade
the misfolded protein first by targeting it
with ubiquitination and finally by
proteolysis.
WHAT ARE MUTATIONS?
Mutation
Error in copying of a sequence of
bases
Occur during replication
Base error can occur during
transcription (non-inheritable error)
Ionizing radiation
Can cause mutations
Ex. X-rays, ultraviolet light, gamma rays
Mutagens
Organic compounds that induce
mutations reacting with DNA
 Radiation and mutagens do not become
mutations because the cell repair
mechanisms such as Nucleotide Excision
Repair (NER)
 Nucleotide Excision Repair (NER)
 Prevent mutations by cutting out damaged
areas and resynthesizing them
 Carcinogens
 Mutagens can cause cancer when introduced
to the body.
 Not all mutations are harmful but some
are beneficial that can enhance the
survival rate of the species
 If Mutations are harmful, results in an
inborn genetic disease.
How and Why Do We Manipulate
DNA?
 Gene manipulation includes gene splicing, use
of recombinant DNA techniques, forming of the
monoclonal antibodies or PCR (polymerase chain
reaction).
 One example of recombinant DNA techniques
begins with certain circula DNA molecules found
in the cells of the bacterium Escherichia coli.
 Plasmids
- consists of double-stranded DNA arraged in
a ring.
 Restriction Endonucleases
-certain highly specific enzymes
that cleaves DNA molecules at specific
locations.
What Is Gene Therapy?
 Gene Therapy
- viruses can be used to alter somatic
cells, where a genetic disease is treated by the
introduction of a gene for a missing protein.
 Adenosine deaminase
- an enzyme involved in purine catabolism
 Severe combined immune deficiency
- individuals who are homozygous for
adenosine deaminase deficiency
- “the bubble boy” syndrome
 Two types of delivery methods in human
gene therapy.
1. Ex Vivo
- a type used to combat SCID
-means that somatic cells are removed from the
patient, altered with the gene therapy, and then
returned to the patient.
- the most common for this approach is the
Maloney murine leukemia virus (MMLV)
 Expression cassette
- contains the gene being administered such as
the ADA gene, along with a suitable promoter.
2. In vivo
- virus is used to directly infect the
patient’s tissues.
 Adenovirus
-most common vector for this delivery
-a particular vector can be chosen
based on specific receptors on the target
tissue.
Gene therapy has been approved in
humans only for the manipulation of
somatic cells.
 What is Epigenetics?
The study of epigenetics is an active area
of research.
 Epigenetics refers to changes in DNA
that are not reflected in the actual base
sequence.
 Epigenetics modifications of DNA act
like switches that turn on or off certain
genes.
The best known example of an epigenetic mechanism
is DNA methylation, where a cytosine is tagged with a
methyl group.