least one corresponding to each of the 20

BIOCHEM: Gene amino acids required for protein synthesis.

Expression part 2 c. Ribosomal RNA (rRNA)–A ribosome is a

nd
March 8, 2018 – Dr. Santos – 2 Semester cytoplasmic nucleoprotein structure that
functions as the machinery for protein
Outline: Recap of Transcription with Post- synthesis from the mRNA templates. On
transcriptional steps, Different types of RNAs the ribosomes, the mRNA and tRNA
Inhibitors of RNA Synthesis, Major Stages in molecules interact to translate into a specific
Translation, Characteristics of tRNA protein molecule information transcribed
Genetic code, DNA Repair from the gene.
It is DNA dependent because transcription requires
the presence of the DNA template.
Recap of Transcription (Part 1 of lecture) RNA polymerase will read the base sequence
present in the DNA of the gene.
Transcription – 2nd process involved in gene
expression; defined as the biosynthesis of RNA The core enzyme of the RNA consist of the two
alpha chains and two beta chains or sub units, plus
In Transcription, RNA copies of selected DNA a regulatory sigma sub-unit.
sequences are made; so what we copy in the DNA
are the selected sequences which are the genes In mammalian cells, there are three kinds of RNA:
a. RNA polymerase I – synthesizes the rRNA
Gene – defined as the segment of DNA that codes b. RNA polymerase II – synthesizes the
for a polypeptide or a protein or an enzyme or an precursor or the mRNA
RNA c. RNA polymerase III – synthesizes tRNA

During Transcription, these are the three kinds of

RNA that we synthesis: (source Lec Guide 1, p301-
302) BASIC STEPS in TRANSCRIPTION

I. Template Bonding
- Involves recognition of specific binding sites
in the DNA template
- The specific binding site is called the
promoter region of the gene or the
promoter site of the gene
- Characteristics of the promoter site, it is rich
in pyrimidine bases
a. Messenger RNA (mRNA) – these are - The pyrimidine bases in DNA are: Thymine
messengers conveying the genetic and Cytosine, and they are the target of
information to the site of protein synthesis, regulatory effectors
where each mRNA serves as a template on - Regulator effectors or modulatorsare
which a specific sequence of amino acids is substances that either stimulate or inhibit
polymerized to form a specific protein gene transcription, the sigma factor is a
molecule, the ultimate gene product. regulatory subunit that enhances the
binding of the core RNA polymerase to the
b. Transfer RNA (tRNA) – act as accessory promoter site of the gene
for the translation of the information in the
sequence of nucleotides of the mRNA into
specific amino acids. There are at least 20
species of tRNA molecules in every cell, at

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 regulate gene transcription specifically
these substances bind to the regulator
signals that
at are present in the coding
strand.

PARTS OF A GENE (found in prokaryotic cells

cells):

 The gene is made up of two strands of

DNA, of which in the:
o Upper strand: the direction is from 5”
to 3”
o Lower strand or Complementary
omplementary
strand –is unti-parallel
parallel to the upper
strand so it runs from 3’ to 5’
direction
 The gene contains the so called template
strand. And, the template strands are  At the left is the transcription start site that is
repeated as the part of the DNA that is where transcription initiation will start. And
shaded the deoxyribonucleotide unit in the
 The template
emplate strand also called Non-coding
Non transcription start site is given a positive
or Anti-sense
sense strand is the DNA strand that number, which is +1.
is transcribed into RNA  All the deoxyribonucleotide units to the right
o So in genes A, B, and D – the of thee transcription start site, constitute the
template strands are found in the transcribed region of the gene. And that is
lower strand that runs in the 3’ to 5’ also the downstream region of the gene (to
direction the right of the transcription start site.
o In genes A, B, and D – there is an  All of the deoxyribonucleotide units to the
arrow that points towards the 5’ end, left of the transcription start site, they are
the reason is that the RNA given a negative (-)) number. So when I say
polymerase reads ds the base -10
10 region, to the left is -1, -2, upto -10
sequence of the DNA template from deoxyribonucleotide units. And this one is
3’ to 5’ the 35th deoxyribonucleotide unit away from
o While for gene C, the template the transcription start site. So this is the
strand is the upper strand and the template strand and to thet left of the
arrow points towards the right, transcription start site is where you’ll find the
because the RNA polymerase
polymera reach promotor site of the gene and that is the
the base sequence in the DNA of upstream region.
gene C from 3’ to 5’ direction
 So the coding strand or also called the
Sense strand is the unsubscribe DNA
strand.. And in the illustration, in gene A, B,
C and D the non-shaded
shaded portion that is the
coding strand.
 So the coding strand is very important
because the regulatory signals are found in
the coding strand. Modulators or effectors
that regulate gene expression and that

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Fig. Gene for Prokaryotes Fig. Gene for Eukaryotes

 So what are these letters? TATAAT Box or  This is another gene which is a tyrosine
Pribnow box,, these are deoxyribonucleotide kinase gene and this is found in eukaryotic
units: cell, this is only an example.
o T is deoxyadenosine  Again, +1 that is the transcription start site.
monophosphate To its right they are the coding region of the
o A is deoxyadenosine gene and to the left they are the promoter
monophosphate site. The deoxyribonucleotide units found
o G is deoxyguanosine monophospate here are present in the promoter region or
 In other words, they are site of the gene.
deoxyribonucleotide units of the DNA. And  So TATA box that provides the ‘where’
they are located in the coding strand of the signal and that confirms fidelity of initiation
gene. The coding strand of the gene is the so that RNA polymerase will start
un-transcribed
transcribed strand of the DNA. transcription right on that part
pa of the gene.
 The deoxyribonucleotide units are the so Because one major difference
di between
called regulatory sequences or regulatory transcription and translation:
signals. And, modulators or effectors o Is that in transcription there is no
regulate gene transcription by binding to the proof-reading
reading mechanism. There is
regulatory signals and (a) if it is a positive no such thing as 3’ exonuclease
modulator it will stimulate gene activity. 3’ exonuclease is the
transcription, (b) if it is a negative modulator enzyme involve in proof-reading
proof the
it will inhibit gene transcription. process of replication.
replication 3’
 The TATAAT Box of prokaryotes is also exonuclease will remove the
called Hogness box. The Hogness box will erroneously inserted
tell the RNA polymerase where the deoxyribonucleotide unit and will
transcription start site is so that the TATAAT insert or put in the correct nucleotide
box is the where signal it will tell the RNA units. There is no such thing as this
polymerase that 10 bases away from the in translation. So it is very important
Pribnow box is the transcription start site. that the RNA polymerase will know
 This regulatory signals on the other hand exactly where the transcription start
will tell the RNA polymerase how many site is.
times will the gene be transcribed,
cribed, once  All of these regulatory signals they
twice or several times. So that is the determine the frequency of transcription
frequency signal, how many signal and that initiation.
is the where signal. They are found within  In eukaryotes, you have the TATA Box
the promoter side of the gene. which is also called Hogness box. box And this

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 CAAT box is the frequency signal, read the base sequence of the DNA
frequency of initiation. template. It is also the DNA
polymerase that will form the
phosphodiester bond that will connect
II. Chain Initiation to deoxyribonucleotides together.
o Wherein you start synthesizing the o DNA template is read byRNA
RNA and there is no need for a polymerase from 3’ to 5’ direction.
primer molecule, you start right away o Direction of elongation is from 5’ to 3’.
synthesizing the RNA. o The RNA is synthesized from 5’ to
1. Alignment of first two NTP’s (Nucleside 3’ direction. And in order
Triphosphate) to their complementary o New nucleotide units are added to 3’
bases on the DNA template. end of growing RNA polymer.
o Now in replication the first part of the o And in order to observe this all
daughter DNA that is formed is the incoming ribonucleotide units will
5’ end, the same in transcription. have to be added to the 3’ end to of
a) 1st NTP (forms the 4’ end) – ATP the growing RNA polymer. And in
or GTP that regard transcription is similar to
o Wherein the first part of the RNA replication. They are the same in
that is formed is also the 5’ end. the matter by which DNA and RNA
And, the first substrate which are is synthesized.
nucleoside triphosphates that align
themselves to the DNA template is
either ATP or GTP. They will be the IV. Termination and Release
sources of adenosine 1. DNA template contains stop signals and
monophosphate or guanosine this is ushered in by a region in the DNA
monophosphate that will form the 5’ that is rich in deoxyribonucleotide units
end of the RNA transfer of guanine and cytosine (GC-rich)
b) 2nd NTP – UTP or CTP followed by an adenine-tyrine (AT-rich)
o The 2nd NTP to be aligned, they are region.
UTP or CTP, which will be the 2. Rho protein is essential for chain
sources of uridine monophosphate terminationin some species, some and
and cytidine monophosphate that not all.
will be attached to either adenosine
monophosphate or guanosine  After that you have already synthesize
monophosphate. mRNA, tRNA, and rRNA.

 So the 5’ end could either be AMP or

GMP and the 2nd deoxyribonucleotide V. Posttranscriptional Modification of
unit is either UMP or CMP. They are RNA (Primarily in Nucleus)
the 5’ end of the RNA already. 1. Addition of nucleotides to 3’ to 5’
 So the first NTP plus RNA polymerase terminal.
will form the initiation process. o The newly synthesized RNA is prone
to degradation by endonucleases
and exonucleases. So you have to
protect the 3’ end and 5’ end so that
III. Chain Elongation they will not be hydrolyzed and
 Let us now elongate the growing RNA degraded by the 3’ exonuclease and
polymer. 5’ exonuclease that are present in
o Sigma factor dissociates from core the nucleus.
RNA polymerase. o So these exonucleases will cleave
o That is important because the core the 5’ nucleotide by cutting the
RNA polymerase is the one that will

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 phosphodiester bond, the same
thing in the other end.
B. Inhibitors that bind to RNA polymerase
a) How do we protect the 5’ end of the o Rifampicin – the most effective anti-TB
newly synthesized RNA?By added a drug in the market today;
Methylated Guanine nucleotide  it inhibits transcription by binding to
called CAP at the 5’ end.
the RNA polymerase, so if there is no
b) The 3’ end is protected by adding
a long stretch of poly A transcription then there is no mRNA
nucleotides that came from that will be synthesized if there is no
adenosine triphosphate. mRNA there will be no template for
protein synthesis, so imbalance
2. Modification of ribose, it may undergo production of protein in the
Methylation reaction where the methyl microorganism may produce a
group will be donated by S-adenosyl- bacteriocidal effect or bacteriostatic
methionine. The enzyme involve is effect
methyl transferase. o Amanitin – toxic principle found in
poisonous mushroom;
3. Modification of bases thereby forming  Amanitin inhibits RNAP II that
peculiar or unusual bases like synthesizes the heterogenous
pseudoridylate, ribothymidylate which
hnRNA, which is the immediate
are not found in mRNA and rRNA, but
they are found only in tRNA. precursor of mature RNA.

4. Nucleolytic and ligation reactions, this

involve removal of introns and splicing REGULATION OF EUKARYOTIC GENE
of exons. TRANSCRIPTION
o Exons are defined as the coding
segments of the gene, while the  Most of the regulation of gene expression
Introns are the intervening occur at the level of transcription.
segments of the gene that do not
code for amino acids. A. Differential expression of genes
o What do you find in exons that are o “Chargaff’s Rule– that the base sequence
not found in introns? Answer: of the DNA that is found in the nucleus of
Codons, they specify the amino the fibroblast is the same base sequence
acid that will be incorporated to the
of DNA that is found in the neutrophils for
polypeptide chain during protein
example, the same DNA that you find the
synthesis
neurons, the same base sequence of
NOTE: Posttranscriptional processing of the DNA that is found in the liver and all
primary transcript is important in the regulation of other organs in the body, but the genes
gene expression. are not expressed equally in all cells of
the body.
o Some of the genes are found in the
INHIBITORS OF RNA SYNTHESIS/TRANSCRIPTION inactive chromatin others are found in the
active chromatin. When you say “active”
A. Inhibition that bind to the DNA template chromatin this is the DNA where the
1. Actinomycin D – antibiotic genes present are actively being
2. Aflatoxin – derived from fungus synthesized and when it happens that the
(Aspergillus flavus); associated with gene may be present in the nucleus but
hepatoma or liver cancer the gene is present in the inactive
chromatin this is part of the DNA that is

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 not being transcribed. So if it so happen o What is the charge of basic amino acids
that if a particular gene is present in an lysine and arginine at physiologic pH?
inactive chromatin it can’t be transcribed, Answer is positive.
translated into a protein molecule. o So Histone being positive binds the
o Example: B-globin gene – it codes for the negatively charged DNA through
beta subunit of globin, and globin electrostatic attraction.
becomes attached to heme to form either o What happens when you add acetyl
myoglobin or hemoglobin or groups to the lysine residues in the amino
cytochromes. So the B-globin
globin gene terminal tails of the histone? Remember
cluster is in the “active” chromatin in the that there are 8 histone molecules at the
reticulocyte which is the mature RBC. In center, so acetylation will reduce the
th
the reticulocyte that is where you’ll positive charge of these tails of the
synthesize hemoglobin, and to do so the histone residues. As a result there will be
globin portion should have the beta decrease binding g affinity of histone to
subunit. Because hemoglobin is made DNA,, if you reduce the number of positive
up of two kinds of polypeptide chains, charge you also reduce the electrostatic
alpha and beta, at least for the adult attraction between DNA and histone so
hemoglobin. But in muscle cells, the you disruptt the nucleosomal structure.
structure
beta globin gene cluster is found in the The DNA will be released from its
inactive chromatin. And that is the interaction with the histone. So when that
reason why we do not synthesize happens regulatory DNA elements
hemoglobin in the skeletal muscle cells, present in the promoter region of the
we synthesize only hemoglobin in the DNA will become more accessible to
reticulocyte cells and the reason is transcription factors
factors.
differential
erential expression of genes.
C. Presence of enhancer and repressor
B. Chromatin remodeling elements
o Example:: Histone acetylation by acetylase o Enhancers are substances that stimulate
– Histone
one is a component of nucleosome gene transcription.
which is the structural and functional unit o Repressor elements are substances that
of the chromatin. The nucleosome is will inhibit transcription.
made up of 8 histone molecules at the o Enhancing binding proteins acting as
center and wrap around it is a segment of transcription activators facilitate binding of
DNA. Histone is a basic protein, it is the basal transcription machinery to the
made up of basic amino acids like glycine promoter region of the DNA.
and arginine.
o What kind of interaction is present
between DNA and Histone? Answer is
Ionic bond or salt bond or electrostatic
bond and it is a bond between positively
charged molecule and a negatively
charged molecule.
o What is the charge
rge of DNA at physiologic
pH of the body?Answer
Answer is Negative
because of the phosphate group which is
negatively charged.

o So you have here a gene, that is the

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 Transcription start site and to its right is o Example: There are 3 unique motifs that
the structural gene that codes for amino exhibit high binding affinity to the correct
acid. To its left, is the promoter region of region of the DNA, to the promoter region
the gene and this is the promoter site of the DNA and these are:
where you have the basal expression. (a) Helix-turn- helix
o You have the TATA Box that confers the (b) Zinc-finger
‘where” signal, where is the transcription (c) Leucine zipper.
start site? o So there motifs enhance the binding of the
o You have the CAAT box, where you have protein to DNA. These proteins are
the frequency signal. actually regulatory proteins and are
o So the TATA box and CAAT box is the usually called transcription factors. So
basal expression site of the promoter proteins provide a ligand binding site for
region of the gene. modulators or effectors.
o These regulatory elements are made up o Again modulators or effectors are
of deoxyribonucleotide units that regulates substances like hormones, metallic ions
gene transcription. And to this regulatory that would either stimulate or inhibit gene
element when a substance bind to these transcription. But there modulators of
regulatory elements, there will either be effectors do not bind directly to the
promotion of gene transcription or regulatory signals or elements that are
inhibition of gene transcription. These present in the coding strand, in the
regulatory elements could either be promoter region of the DNA.
enhancers and silencers. So when a o So what binds directly to these regulatory
hormone binds to a hormone response elements? They are the regulatory
element and the hormone binds to the proteins, they are the transcription factors.
enhancer region that will stimulate gene o So how will metallic ions regulate gene
transcription. expression?They bind to the protein and
o The same thing is true for some metallic the protein binds to the DNA. And by
ions, some metallic ions serve as silencer doing so the hormone or the metallic ion
and when they bind to the silencer may induce a conformational change on
element of the gene, they will inhibit gene the regulatory protein and somehow that
transcription. will affect transcription.

E. Other modes of regulation

D. Regulatory proteins having special motif
bind with high affinity to the correct region  Gene amplification – it explains how some
of the DNA. people develop toleranceto certain drugs; such
o Motifs are super secondary structures of a as the drug resistance to methotrexate by
protein molecule. increasing the number of dihydrofolate
o Remember the core levels of structural reductase.
organization of a protein (1) primary – is o Increase in the number of genes available
sequence of amino acids or number of for transcription results in increase in the
amino acids, number of peptide bonds; (2) number of RNA and proteins. This is
secondary – there are 2 basic secondary generated by a process of repeated
structures which are alpha helix and beta initiations during DNA synthesis thereby
sheet, (3) tertiary and (4) quaternary. providing multiple sites for gene
o Motifs are different combinations of alpha transcription.
helix and beta sheets.

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 So you rearrange introns and exons,
particularly exons.

THIRD STEP: TRANSLATION

o Translation or Ribosomal Protein

Synthesis.– occurs in the Ribosome
o Let’s say that this is a gene, it is made up of
2 exons and this gene when transcribed o Translation – is the process by which a
protein is synthesized from the information
and translated later on will be expressed
contained in a molecule of messenger RNA
in the form of an enzyme.
o So in the unamplified gene, there is only mRNA).
one gene. o During translation, an mRNA sequence is
read using the genetic code, which is a set
o In the amplified gene, you have several
of rules that defines how an mRNA
copies of the same gene (7 copies in the
example), 7 genes transcribed and sequence is to be translated into the 20-
20
translated to 7 enzymes. letter code of amino acids.
o So let us say that a drug at a given dose
inhibits only 1 enzyme. And in the
FIVE
\ MAJOR STAGES OF TRANSLATION
TRAN
amplified gene you give the same dose of
the drug it will inhibit
hibit only one enzyme so
the rest, 6 of the other enzymes are not I. Activation of Amino Acid
inhibited. o It involves the formation of aminoacyl-tRNA
aminoacyl
o Example is methotrexate which is a complex
chemotherapeutic drug. o Catalyzed by a highly specific aminoacyl-
aminoacyl
tRNA synthetase
 This 2nd statement is very important
because you do not just attach an amino
 Gene Rearrangement – This is the process
acid to any transfer RNA because the
involved in the synthesis of different antibodies
tRNA is the carrier of the amino acid.
acid
by lymphocytes. It allows the generation of 109
 Andnd the tRNA contains the anticodon
– 1011 different immunoglobulins from a single
which during translation or protein
gene. The variable region of the antibody is the
synthesis should match the codon that is
result of somatic recombination of segments
egments
present in the mRNA that serves as a
within both the light and the heavy-chain
chain genes.
template for protein synthesis.
o You have learned that a lymphocyte can
 And during protein synthesis, the correct
produce a great variety of antibodies,
amino acid should be incorporated as the
which are immunoglobulins.
protein or polypeptide is being
o In the immunoglobulin genes you have
synthesized and elongated.
different introns and exons. So you can
 What dictates the sequence of the ami amino
have a combination
ation of introns and exons
acid that will be incorporated to the
and because of that you can produce a
polypeptide chain?It
chain? is the genetic code.
great number of antibodies. But only one
And the original genetic code is present
antibody is specific against one antigen.
in the DNA.
o How are you able to produce this great
 What does the DNA contain?Answer:
contain?
variety of immunoglobulins or
genes. What are genes? They are
antibodies?Through
Through gene rearrangement.
rearrangemen

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 segments of DNA that code for a E. Coli Ribosomal Structure:
polypeptide, a protein, or an enzyme.
 So during protein synthesis, you do not  We are going to use the prokaryotic ribosome
just insert or incorporate amino acids at rather than eukaryotic ribosome because it is
random. There has to be something that simpler and we are just after the process of
translation, so whether it is prokaryotic or
dictates it, and that is the DNA genetic
eukaryotic translation the process is the same.
information that is encoded in the DNA
which is transcribed into mRNA. So the o E.Coli ribosome is a 70s (refers to the
mRNA that serves as a template for sedimentation rate) ribosome which is inactive,
protein synthesis is a copy of the gene in must first dissociate into their 30S and 50S
the DNA. And so the amino acid should subunits (‘S’ gives an idea as to the size and
be incorporated to the correct tRNA molecular weight of a given protein)
which should have the correct anticodon
that should match the codon that is o Initiating f-met-tRNA can bind only to P site (or
present in the mRNA. So the enzyme Peptidine site); exception to the rule because
other incoming aminoacyl-tRNA complex bind first
should be highly specific. – This is an
to A site (or Aminoacyl site)
endergonic reaction because you need
ATP to incorporate the amino acid to the
correct tRNA. o Translation of codon of mRNA is from 5’ to 3’
direction – this what makes translation
different from replication and transcription
Characteristics of transfer RNA:  Because we replicate and transcribe the DNA
1. tRNA is an adapter molecule with several parts template from 3’ to 5’ direction, whereas the
a. Amino acid arm – binds the amino acid mRNA is translated (read the base sequence)
b. Anticodon arm – recognizes the codon in mRNA from 5’ to 3’ direction.
c. Enzyme recognition site  In replication and transcription, the first part of
d. Ribosome attachment site the daughter DNA that is formed or the first
part of the RNA transcript that is formed is the
5’ end.
2. tRNA has high content of peculiar bases – these
peculiar bases are not found in mRNA and rRNA  In translation, the first part of the polypeptide
o e.g. inosine, dihydrouridine, pseudouridine and or protein molecule that is formed is the N-
terminal amino acid.
methylated bases
st
3. tRNA is depeated as having a Cloverleaf o N-terminal amino acid is 1 part of protein to be
appearance but in actual x-ray crystallography formed
study, tRNA looks more like a twisted letter “L”  How do we elongate the protein molecule?
From the N-terminal to the C-terminal.

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II.Initiation of the polypeptide chain  mRNA is derived from heterogenous
nuclear RNA (hnRNA) in eukaryotes
1. Binding of mRNA to the smaller subunit  hnRNA contains introns and exons
(30S) of ribosome.  During the transformation of hnRNA to
mRNA you remove the introns because
2. Formation of initiation complex – aminoacyl- they do not code for amino acids, hence
tRNA base pairs with corresponding codons would only need the exons
on the mRNA
- Continuation ofInitiation
Initiation of polypeptide chain:
chain
3. Initiating codon in mRNA is AUG
(Adenosine monophosphate, Urasyl
monophosphate, and Guanosine 4. Other requirements during initiation are: GTP
monophosphate) because the sugar is (provide the energy), (Initiation Factors) IF-
IF
ribose not deoxyribose; the initiating amino 1, IF-2, and IF-3,
3, Mg
acid that corresponds to AUG is
methionine (eukaryotes and mammalian 5. Attachment of large ribosomal unit (50S)
cells) while N-formylmethionine
formylmethionine is the
initiating amino acid for prokaryotes 6. Inhibited by streptomycin
o One good thing about gene expression is
that it will should you the sites of action of
the different antibiotics. On how
antibiotics produce bactericidal or killing
effects on the microorganism or a
bacteriostatic effect as it inhibits the
growth and duplication of the
microorganism.
o So the initiation stage of protein synthesis
is inhibited by streptomycin.

 Animation of Initiation Stage of Translation:

Translation

o This is a diagrammatic illustration of the o That is the mRNA which has the ribosome
mRNA. binding site, AUG is the initiating codon,
o SD - Shine Delgado Sequence which is the and there is small ribosomal unit and the
ribosome binding site. large
ge ribosomal subunit. So what
o Reorientation: happens is that the small ribosomal unit
 On the left is the 5’ end where you have binds to the ribosomal binding site in the
CAP which is Methylated Guanine mRNA.
Nucleotide o The large ribosomal subunit will bind to
 On the right is the 3’ end because of the the small subunit, there by forming a
presence of the poly-A A tail that protects complete ribosome.
the 3’ end of the RNA transcript o So a complete ribosome is made up of
 Initiation Codon: AUG – contained within small unit plus large ribosomal subunit.
the Kozal sequence in eukaryotes o The large ribosomal unit contains the P
 Stop Codons:: UGA, UAG, UAA (peptidine) site and the A (aminoacyl)
 PAS – Polyadenylation signal which site.
comes after the stop codons.

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 o The initiating methionine or F- peptidebond is Peptidyl transferase,
formylmethionine tRNA complex, so this and it is inhibited by Chlorampherincol.
is the tRNA that contains the anticodon
and that is the AUG which is the o Catalyze by peptidyl transferase
initiating codon in the mRNA. o Enzyme is inhibited by chloramphenicol
o Product is dipeptidyl-tRNA bound to A
site
III. Elongation of polypeptide chain – starts when
initiating complex is bound to the P site
3. Translocation
o Initiating Complex – methionine tRNA
complex bond to the P site or a) Ribosome moves to the next codon of
formylmethionine tRNA complex bound mRNA
to the P site
b) Peptidyl-tRNA shifted from A site to P site
Three major steps: (that takes place one after the c) Requires EF-G and GTP (for the shifting
other, continuously until you completely from A to P site)
synthesize the polypeptide or protein)
d) Inhibited by erythromycin
1. Binding of incoming aminoacyl-tRNA
complex
a) Attaches to the A site – because the P
site is already occupied by the initiating
complete IV. Termination of polypeptide chain
b) Requires elongation factors (EF-T and 1. Presence of termination signals in mRNA
EF-G) and GTP will provide the energy 2. UAQ, UAA and UGA are the stop codons
c) Blocked by tetracycline which a broad or nonsense triplets; do not code for any
spectrum antibiotic (inhibited) amino acid
3. Requires releasing factor (RF’s 1, 2 and 3)
2.Peptide bond formation  Hydrolytic cleavage of polypeptide
 Release of empty tRNA from P site
o In replication and transcription the kind  Dissociation of 70S ribosomal unit
of bond produced between two
deoxyribonucleotide units is
phosphodiester bond.  Animation of Elongation and Termination:
 In replication, the major enzyme o The ribosome moves to the initiating
involved in the formation of codon which is AUG.
phosphodiester bond is DNA o Methionine or formylmethionine tRNA
polymerase. complex matches the initiating codon on
 In transcription, the major enzyme the mRNA following the base pairing rule
involved in the formation of  Adenine paired w/ Urasyl
phosphodiester bond is RNA  Urasyl paired w/ Adenine
polymerase.  Guanine paired w/ Cytosine
o In translation the bond formed between o In what part of the ribosome does that
two amino acid residues is peptide take place?Answer is P site, because
bond. Methionine or Formylmethionine tRNA
 Intranslation, the major enzyme complex binds only to the P site.
involved in the formation of o The next aminoacyly tRNA complex with
different anticodon and aminoacyl come

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 into the picture. What happens is that in the polypeptide the principle of protein
the 2nd transfer RNA binds to the A site synthesis is the same.
of the large ribosomal subunit and o Termination occur when the stop codon
matches the next codon. The codon occupies the A site.
next to AUG which happens to be UCU.
And the anticodon is AGA. – This is the
first step during elongation which is the Other name for the Stop codons:
binding of the incoming aminoacyl tRNA
complex to the enzyme. o UGA - Opal codon
o UAG –Amber codon
o Peptide bond formation. Methionine or
o UAA - Ochre codon
Formylmethionine tRNA complex will be
detached from the first tRNA and will be  Stop codons were historically given many
attached to 2nd amino acid via a peptide different names, as they each
bond, catalyzed by peptidyl transferase. corresponded to a distinct class of mutants
o Next is translocation, the ribosome moves that all behaved in a similar manner.
to the next codon so the di-peptidyl These mutants were first isolated
tRNA complex is shifted from the A site within bacteriophages (T4 and lambda), vir
to the P site. – This completes one uses that infect the bacteria Escherichia
round of elongation. coli. Mutations in viral genes weakened
o The next round is the 3rd aminoacyl tRNA their infectious ability, sometimes creating
complex come into the picture: (1) viruses that were able to infect and grow
binding of the incoming tRNA complex within only certain varieties of E. coli.
to the A site, (2) peptide bond formation,
so the di peptide will be detached from o No tRNA has the anticodon or stop codon.
the 2nd tRNA and through a peptide A release factor recognizes these stop
bond it will be connected to the amino codons. And the release factor attaches
acid bound to the tRNA which in turn is to the A site and breaks up the complex,
attached to the enzyme, and (3) which is the newly synthesized protein
translocation. or the last tRNA and the ribosomal
o The ribosome moves to the next codon subunit dissociate.
and the tri peptidyl tRNA complex
shifted from the A site to the P site.
o Then UAA which is the stop codon
appear. So as the ribosome moves to
the next codon in the mRNA it encounter V. Posttranslational processing
the sop codon (UAA) which does not
1. N-terminal and C-terminal modification
code for any amino acid.
o E.g. removal of formyl group from f-met by
o So the complete polypeptide which is
deformylase
actually not a polypeptide but only a tri-
peptide is released. One example of tri-
polypeptide is Glutathione.
o The tRNA is released, the complete 2. Loss of signaling sequences – proteins that
ribosomal subunit dissociate to small are destined to be components of the cell
and large ribosomal subunit. You membrane they are synthesized with a
synthesize a tri-peptide. signal peptide; proteins that are destined to
o Same event will take place even if you exported out of the cell to go to the other
synthesize a larger protein molecule, no organs of the body they are synthesized
matter how many amino acids there are with a leader peptide.

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 o ‘Leader’ peptide these
hese are extra
extr amino
acid resides found in the N-terminal
terminal end
of polypeptide
o This “leader” peptide directs the protein to
its ultimate destination in the cell,
cell which
is the lumen or cisternae of the rough
endoplasmic reticulum
o ‘Leader’ peptide are removed by specific
peptidases in the smooth endoplasmic
reticulum

3. Phosphorylation of hydroxyl amino acids

(Ser, Thr and Tyr) – they
hey are found in the
active site of covalently modulated
regulatory enzymes which are activated or
inactivated through phosphorylation or
dephosphorylation reaction

4. Carboxylation reactions – like the o These are the list of inhibitors of gene
maturation of the clotting factors such as expression, and there mechanisms of
prothrombin in which glutamic acid residues action.
are carboxylated, they undergo gamma
carboxylation GENETIC CODE
5. Methylation of R groups – by methyl
transferases, the donor of the methyl group
o There are 20 different amino acids compose
is S adenosylmethionine
of protein molecule
6. Attachment of carbohydrate side chains – in
o There are 4 code letters in DNA (A, T, G and
the hydroxyl amino acids via an O glycosidic
C), which are the nitrogenous bases found
bond or you can attach the sugars to an
in DNA
amino group of asparagine, glutamine and
o Three nucleotide residues of DNA are
lysine through the glycosylation reaction
required to code for each amino acid
7. Addition of prosthetic groups – like the
(codon),
), codon therefore is a triplet because
addition of heme to globin
42 = 16 (insufficient to cover all 20 amino
8. Formation of disulfide cross links–
links between
acids)
two
wo adjacent cysteine amino acids
43 = 64 codons (more than enough for the
20 amino acids)
o All of these processes happen after the
completion of the formation or synthesis of a
polypeptide or protein which is called the
posttranslational stage. CHARACTERISTICS OF THE GENETIC CODE

1. The genetic code is universal.

o The amino acid code words
wor are identical in all
species, whether prokaryote or eukaryote.

2. The genetic code is commaless.

o No punctuation or signal is required to
indicate the end of one codon and the

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 beginning of the next. That is the basis of
“frameship mutation” DNA DAMAGE
Source: Lecture Guide_2 –MECHANISM
and REPAIR page 284-288
 There are 2 kinds of frameship mutation:
deletion (meaning you delete a o The DNA sequence can be changed
nucleotide out of the 3 nucleotide bases as the result of copying errors
it will only be 2 which should not be the introduced by DNA polymerases
case because the codon would need 3 during replication and by
hence the genetic frame will adjust to environmental agents such as
the left and pronounce an entirely mutagenic chemicals and certain
different sequence of codons and when types of radiation.
translated you may have carbon o Repair of DNA damage is important
sequence of amino acid) and insertion for maintaining genomic integrity and
frameship mutation (means inserting a in preventing propagation of
nucleotide there will be 4 nucleotides in mutations horizontally so much so
the codon which should not be the case that somatic cells could no longer
as well which will cause for the genetic function and undergo apoptosis.
frame to move to the right) – different
set of codons you are going to produce TYPES OF DNA DAMAGE
an entirely different protein or
polypeptide 1. Single-base alteration caused by depurination,
deamination of cytosine to uracil and adenine to
3. The genetic code is non-overlapping hypoxanthine, alkylation of base, insertion or
o Each group of 3 bases (codon) specifies deletion of nucleotide and base analog
only one amino acid. incorporation.

4. The genetic code is degenerate 2. Two-base alteration caused by UV light-induced

o Different amino acids have different thymine-thymine dimer and bifunctional
number of codons, 43 = 64 codons: alkylating agent cross-linkage.
o E.g. leucine and serine = 6 codons
3. Chain breaks caused by ionizing radiation,
Histidine and tyrosine = 2 codons
radioactive disintegration of backbone element
Tryptophan and methionine = 1 codon and oxidative free radicals.

-AUG the only codon for methionine 4. Cross-linkage between bases in the same or
 When 2 different amino acids have code opposite strands and between DNA and protein
words in which the 1st 2 bases are identical, molecules.
the 3rd base of one is filled only be purines MECHANISMS OF DNA REPAIR
and the 3rd base of the other only by
pyrimidines.
o E.g. histidine = CAU 1. Mismatch repair (MMR)
CAC pyrimidines 2. Base excision repair (BER) removes small, non-
helix-distorting base lesions from the genome
Glutamine = CAA 3. Nucleotide excision repair (NER) removes bulky
CAG purines helix-distorting lesions
4. Homologous recombination (HR) and non-
o “Wobble” allows some tRNA to homologous end-joining repair (NHEJ)
recognize more than one codon

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