Gene Expression

• Longest part of the cell cycle

Replication 1. Responsible for variation in cell cycle in different organisms
DNA o 8 hours to > 100 days
\ 2. Cell’s irreversible decision to proliferate is made here
o Aided by Cell Division Cycle Proteins (CDC) which helps cell to
get beyond threshold → “Restriction Point”
Transcription Reverse Transcription o CDCs are protein kinases
→ Kinases are phosphorylating enzymes
3. Cells may enter a quiescent phase (G0)
o Cells do not divide (e.g., neurons and muscle cells)
RNA o They will stop multiplying when they reach maturity
Protein → Important in cases of stroke (cerebral thrombosis) due to
Translation
lack of blood supply
→ Stroke = paralyzed (when severe, impairment of
• It provides the basic framework on how genetic information flows from a movement of the body can be permanent
DNA sequence to a protein product inside the cell
• This process of genetic information flowing from DNA to RNA to Protein is
called Gene Expression • Short supply of nutrients
• “Contact Inhibition”
The Three Processes involved are: o When cells are in close proximity with one another (crowded), they
o Replication and Transcription – which takes place in the nucleus will stop multiplying (seen in normal cells)
o Translation which takes place in the cytoplasmic ribosome o EXCEPTION: proliferation will not stop in cancer cells causing
tumors because they have lost the property of “contact inhibition”
• In Replication the information encoded in the parent DNA is copied by 2
daughter DNAs
o Biosynthesis of DNA • Only period when DNA is synthesized
• In Transcription, the genetic information in the DNA is copied and passed • DNA Replication precede actual cell division during mitosis
on to a messenger RNA (mRNA) o When you inhibit DNA Replication, there is no cell division
• mRNA now carries this information from the nucleus to the ribosome o Parent cell must subdivide into 2 daughter cells (needs to have their
where it serves as a template for Translation, which is Protein Synthesis own daughter DNA)
• In the figure above, you will notice a broken arrow from RNA to DNA. That o This is one principle of chemotherapy using drugs to kill cancer cells.
arrow signifies Reverse Transcription, catalyzed by Reverse Transcriptase This will target the S stage of eukaryotic cell cycle, inhibit DNA
o This allows the reverse transcribing RNA viruses such as retroviruses Replication
use the enzyme to reverse transcribe their RNA genomes into DNA,
which is then integrated into the host genome and replicated along
with it o Carcinogens
o This forces the cell to make more viruses o Surgical removal of tumor
o Mitogens – proteins that bind to cell surface receptors and induce
The Central Dogma is usually a one-way process, from replication to cell division
transcription, and from transcription to translation. So, there is no way that you
can synthesize protein directly from DNA as the template.
• Cells are prepared for mitosis
• Promoted by cyclin, a protein that triggers entry of cells into M Phase

• M (Mitotic) Phase
• G1 (Gap) Phase
• S (Synthetic) Phase
• G2 Phase

• G1 – commitment to DNA Replication

• S – DNA Replication
• G2 – preparation for Mitosis
• M – Mitosis and Cell Division
• Cell division occur • G0 – Quiescence
• Parent cell will subdivide into two daughter cells

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 Before you can have cell division (M Phase), you must have first DNA
Replication (S Phase). So, DNA Replication precedes cell division, which
means that without DNA Replication, cell division will not take place. This is
the reason why many chemotherapeutic agents or drugs that treat cancer,
their MOA targets the S Phase of the cell cycle. If these drugs can inhibit
DNA Replication, then cancer cells cannot subdivide because in order for
the 2 daughter cells to survive, it must have its own set of DNAs. In DNA
Replication, the parent DNA subdivides into 2 daughter DNAs.
• Ori Site is the origin site of replication
• Involves association of sequence-specific double-stranded DNA (dsDNA)
binding protein to DNA
• Theoretically, this may happen, o E.g., DNA A (E. Coli)
but in reality, it does not happen O Protein (Bacteriophage)
• One daughter DNA conserves the ORC (Yeast)
parental DNA while the other • The dsDNA-binding protein bind to a 150-250 base pair DNA sequences
daughter DNA gets the newly adjacent to a region rich in Adenine and Thymine (A & T)
synthesized DNA
• In other words, the parental DNA, Question: Why Adenine & Thymine rich regions?
after Replication, becomes a Answer: Because these A & T regions separates/unwinds easily. A & T have
daughter DNA and the daughter 2 hydrogen bonds while C & G have 3 hydrogen bonds. 2 hydrogen bonds
DNA gets the newly synthesized are easier to separate/unwind than 3 hydrogen bonds which is why A & T
DNA rich regions is where the Ori site can be found.

Recall Base-Pairing Rule:

For DNA: A pairs with T, C pairs with G
For RNA: A pairs with U, C pairs with G

1. Replication origin are unique DNA segments that contain multiple

short repeated sequences
2. These short repeat units are recognized by multimeric origin-binding
• This is the type of replication in proteins
living organisms 3. The Ori Site is adjacent to a region of the DNA rich in Adenine and
• Each daughter DNA conserves Thymine
one strand of parental DNA and
one stands of newly synthesized
DNA
• Formation of Protein-Protein and Protein-DNA interactions
• 2 Daughter DNAs: one strand
• Helicase unwinds short segments of DNA
from parent DNA and the other
o Unwinding is an expensive process
strand from newly synthesized
o Helicase hydrolyzes ATP – endergonic process
DNA
o E. coli uses a complex of DNA B Helicase and DNA C Protein
→ DNA B Helicase – protein-protein interaction
→ DNA C Protein – protein-DNA interaction
→ In E. coli, it is a complex of DNA B Helicase and DNA C Protein that
serves this function
• Single-Stranded Binding Proteins (SSBs) prevent reannealing
(reattachment) of separated strands
• Requires DNA Topoisomerases to relieve supercoils formed during
Question: Why is it automatic that in living organisms, they have unwinding
Semiconservative Replication? o Helicase opens up the DNA at the Replication Fork while the SSBs coat
Answer: It’s because it is in the very nature of the structure of the DNA. The the DNA at the Replication Fork to prevent reannealing and rewinding
DNA is a duplex made up of 2 strands of Deoxyribonucleic Units connected of the DNA
to one another by phosphodiester bonds. During Replication, the DNA o The winding problem of DNA arises due to the intertwined nature of
duplex separates into 2 single strands of DNA (2 separate parent strands). each double helical structure
Each single parent strand now serves as a template for the biosynthesis of o During DNA Replication, DNA becomes overwound ahead of the
the new daughter DNA. Parent Strads run in a 5’ to 3’ direction while the Replication Fork
Newly Synthesized Strands run in a 3’ to 5’ direction. This shows that they o A double helix that has undergone additional twisting is called
are anti-parallel to each other. This is how Semiconservative Replication Superhelix
happens. o Topoisomerase works at the region ahead of the Replication Fork to
prevent supercoiling
o This enzyme prevents the DNA double helix ahead of the Replication
Fork from getting too tightly wound as the DNA is opened up
1. Identification of the Ori Site o If you have a superhelix, helicase may have a hard time unwinding that
2. Unwinding of the DNA segment of DNA
3. Formation of the Replication Fork o Prevents the rotation of unwound DNA in high speeds to prevent
4. Elongation formation of supercoils
5. Removal of RNA Primer
6. Splicing the Okazaki Fragment
7. Proofreading and Correction of Errors

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 • DNA Topoisomerase I – cut a single strand of the duplex nucleases due to the presence of SSBs. Also, SSBs prevent the formation of
o Has a nuclease (strand-cutting) and ligase (strand- secondary structure such as the “hairpin loop”
resealing) catalytic activities, hence, it is called “Nicking-
Resealing Enzyme” What is the problem when there is a hairpin loop?
• DNA Topoisomerase II – nicks/cuts both strands of the DNA When there is a DNA polymerase, it will start to synthesize a daughter DNA.
duplex in order to manage DNA tangles and supercoils But with the presence of hairpin loop, the DNA polymerase will have
o ATP-requiring process difficulty in reading the base-sequence of the DNA template, hindering the
o Etoposide, an anti-cancer drug, inhibits human DNA biosynthesis of the daughter DNA
topoisomerase II
o Also called DNA Gyrase (in bacteria)
o Site of inhibition by quinolones • Branch point in a Replication Eye at which DNA synthesis occurs
→ Inhibited DNA gyrase = no DNA replication = • There are 2 Replication Forks in a Replication Eye or Bubble since DNA
bacteria will be killed synthesis is bidirectional
• Formed by a sequence of 4 events:
• Twists the duplex in the direction which favors unwinding a. Helicase (orange triangle) unwinds the parental DNA
• DNA Gyrase (DNA Topoisomerase II) in bacteria is inhibited by quinolones,
novobiocin, and oxolinic acid

b. Primase initiates the synthesis of RNA Primer (green arrow) that is

essential to start DNA synthesis

• Supercoils have more turn of the helix than relaxed or normal DNA.
• Positive Supercoils interferes with further unwinding of the DNA double
helix.
• Negative Supercoils are those containing fewer turns of the helix
c. DNA Polymerase initiates synthesis of nascent daughter strand
What is the bond that the DNA topoisomerase breaks? Phosphodiester (purple arrows)
bonds of the duplex.

DNA Topoisomerase Introduces a “nick” or a “break” in one or both

strands of the unwinding double helix (swivel
function)
Prevents the entire duplex ahead of the
replication fork from rotating at a high speed
Reseals the nick without requiring energy using
the Nicking-Resealing Enzyme
Twists the duple in the direction which favors
unwinding
DNA gyrase in bacteria is inhibited by quinolones, Both strands of the DNA template are copied simultaneously as the
exemplified by ofloxacin, novobiocin, and replication eye/bubble enlarges
oxalinic acid o Each end of the bubble represents a growing fork where both
Helicase A DNA B Protein new strands are synthesized
Enzyme that unwinds short segments of DNA by
translocating along the lagging strand template
Hydrolyzes ATP; unwinding requires ATP
(endergonic reaction)
Single-Strand Binding Prevent the separated strands from coming
Protein (SSB) together (reannealing) again d. SSBs (yellow circles) bind to single-strand DNA (ssDNA)
Protect single-stranded (ssDNA) from being o This prevents reannealing of the separated strands of the
degraded by nucleases dsDNA
Prevent ssDNA from forming secondary
structures such as a hairpin loop
• Requires priming by a short length of RNA (RNA Primer) (10-200
nucleotides long) complementary to the DNA template
The DNA is made up of 2 strands. These 2 strands are “hugging each other”
• Recall that DNA has 2 anti-parallel strands, one from 3’ to 5’ and the other
because they protect one-another from being attacked by nucleases. They
from 5’ to 3’
conceal their phosphodiester bonds by “hugging each other.” Nucleases
o Leading Strand
such as endonucleases are enzymes that will hydrolyze the interior
→ Read from 3’ to 5’
phosphodiester bond, thereby fragmenting the nucleic acids of the DNA.
→ Only one RNA primer
Because of the fact that the DNA is a duplex, the phosphodiester bonds are
o Lagging Strand
not readily attacked by these nucleases.
During replication, the duplex becomes ssDNA (separated from each other). → Read from 3’ to 5’
Since they are separated from each other, their internal phosphodiester → Replicated discontinuously
bonds are exposed. Still, these bonds cannot be readily attacked by → Away from the replication fork
→ One primer in each Okazaki fragment

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• Direction of synthesis of the RNA Primer is to 5’ to 3’ direction → Forms the Phosphodiester bond that links 2 nucleotides togetehr
• DNA template is read from 3’ to 5’ direction → Involved in Nick Translation
→ Nick Translation – tagging technique where Pol I is used to
We start reading at age 3-5, we replicate at an older age (5) to produce replace some nucleotides of a DNA sequence with their labelled
a younger one (3). So, reading (3’ to 5’), replicate (5’ to 3’) analogues, creating a tagged DNA sequence
• The kind of nucleotide added is dictated by the base-sequence of the o 3’ to 5’ Exonuclease
template strand following the base-pairing rule → Allows Pol I to edit its mistakes (proofreading mechanism)
o A=T/U, C=G (Chargaff’s Rule) o 5’ to 3’ Exonuclease
• The substrates used for the synthesis of the RNA Primers: ATP, UTP, CTP, → Removes the RNA Primer
and GTP • Functions in the repair of damaged DNA through its 3’-exonuclease activity
and polymerase mode
• Serves to connect Okazaki Fragments by deleting RNA Primers and
• Each strand of the DNA duplex serves as a template replacing the strand with DNA
• DNA Polymerase is the principal enzyme involved in elongation • Klenow Fragment
o Reads the base sequence of the DNA template in a 3’ to 5’ direction o Large fragment containing both the polymerase and 3’-exonuclease
o Elongates the daughter DNA from 5’ to 3’ direction (anti-parallel to the activities
strand being copied)
o Adds new nucleotide units to the 3’ end of the primer strand • Participates in DNA repair
• Has 3’-exonuclease activity only

• Major DNA Replicase

• Leading Strand Replicase
• Has 10-subunit complex
• Core Subunit consists of:
The primer (green) is being added with new nucleotide units (purple o Alpha (α) – polymerase activity
arrow) at its 3’ end by the DNA Polymerase o Epsilon (ε) – 3’-exonuclease activity
o Beta (β)
→ Accounts for the high stability of the complex
→ Provides optimum processivity for Pol III

Processivity – ability of the DNA Polymerase to add new

nucleotide units to the growing DNA Polymer
o Number of nucleotides added before a Polymerase
dissociates
• Leading Strand is repeated continuously o DNA Polymerase differ greatly in processivity
o The strand that is being copied towards the direction of the advancing o Some add just few nucleotides before dissociating,
replication fork others add many thousands
• Lagging Strand is repeated discontinuously
o Forms the Okazaki Fragments → Synthesizes leading strand and lagging strand
✓ Synthesis of the leading strand begins with the synthesis
During the biosynthesis of Okazaki Fragments, there must be an of a short RNA Primer at the Ori Site catalyzed by Primase
RNA Primer (green) which is synthesized by primase. DNA (DNA G Protein)
Polymerase adds new nucleotide units (purple arrow) which now → Primosome made up of DNA G Protein (Primase) and DNA B
forms the Okazaki Fragments Protein) Helicase
• Pol III uses one of its core polymerases to synthesize the leading strand
• Eukaryotic DNA has many Ori Sites while another set of core subunit cycles from one Okazaki Fragments to the
• DNA Polymerase III cannot initiate Replication, it requires DNA template next to synthesize the laggings strand
and an RNA primer
Prokaryotic DNA Polymerase Function/Activity
DNA Polymerase I Polymerase Activity
3’ to 5’ exonuclease activity
• Is the major enzyme in Replication 5’ to 3’ exonuclease activity
• Properties: DNA Polymerase II 3’ to 5’ exonuclease activity
o Chain elongation DNA Polymerase III Alpha – polymerase activity
→ Rate at which Polymerization occurs Epsilon – 3’-exonuclease activity
o Processivity Theta
→ Average number of nucleotides added to the daughter DNA being Beta – accounts for high stability of the
synthesized every time the enzyme associates with the Template complex
Strand
o Proofreading
→ This functions to identify copying errors and correcting them • At least 3 DNA Polymerases are required for Eukaryotic Genome
→ This is the Proofreading Mechanism during Replication Replication
• These different polymerases function at the Replication Fork of the DNA
Strands
• Lagging Strand Replicase
• Prototype; first to be discovered by Kornberg in 1957
• Has 3 enzymatic activities: • DNA Polymerase Alpha (Pol α) (>250 kD)
o Polymerase o Initiates DNA Synthesis on both the leading and lagging strand by
→ Gap-filling and synthesis of lagging strand synthesizing a DNA-RNA Hybrid Primer

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 o The replicative DNA Polymerases (Pol δ and Pol ε) then extends DNA
Synthesis from the primer
o Replicates by extending a primer in 5’ to 3’ direction
o Has a tightly associated primase activity
o lacks exonuclease activity
• DNA Polymerase Delta (Pol δ) (170 kD)
o Involved in DNA Replication and Repair
o Mainly involved in the synthesis of the lagging strand
o Lacks primase activity
o Exhibits a proofreading (3’-exonuclease) activity
o Eukaryotic lagging strand synthesis
o DNA Pol I counterpart
• DNA Polymerase Epsilon (Pol ε) (256 kD)
o Highly processive • As shown on the upper picture, the primers (green) initiate DNA synthesis.
o Leading strand synthesis • Once their purpose has been served, they have to be taken out and
o One of the well-established functions for Proliferating Cell Nuclear replaced with an actual DNA.
Antigen (PCNA) is its role in processivity factor for DNA Polymerase • Now focus at the 5’ end of the primer. It will be digested by DNA
Delta and Epsilon Polymerase I through its 5’ to 3’ exonuclease function.
→ PCNA serves as a DNA Clump that ties the polymerase catalytic • One of the functions of DNA Pol I is to take out primers by digesting the
unit to the DNA template for rapid and processive DNA primers at its 5’ end.
synthesis • As shown in the lower picture, we have a huge gap devoid of any primer or
o (+) 3’-exonuclease activity DNA. We need to fill this particular area now with DNA.
o Participants in DNA repair • DNA Polymerase I will fill up these gaps with actual DNA (purple arrow
o DNA Pol III counterpart extending). Even after the primers are taken out, the Okazaki fragments
• DNA Polymerase Beta β (36-39 kD) are still not bonded to each other. It is still not a single continuous strand
o Participates in DNA repair required for DNA maintenance, replication,
recombination, and drug resistance

• DNA Polymerase Gamma (Pol γ) (160-300 kD)

o Replicates mitochondrial DNA
o In X Chromosome of mother

Eukaryotic DNA Polymerase Location Function/Activity

DNA Polymerase alpha Nucleus Primase activity
DNA Polymerase delta Nucleus 3’-exonuclease activity
• Done by DNA ligase which connects the Okazaki fragments to the lagging
Lagging Strand Synthesis
strand of the growing daughter DNA
DNA Polymerase epsilon Nucleus 3’-exonucleae activity
• As shown in the picture above, the Okazaki Fragment has been bonded
Participates in DNA repair
together to become a single strand without any breaks
Leading Strand Synthesis
DNA Polymerase beta Nucleus Participates in DNA repair
DNA Polymerase gamma Mitochondria Replicates mitochondrial
• Short sequences of DNA nucleotides
DNA
• Approximately 150-200 base pairs long
• Synthesized discontinuously and later linked together by the enzyme
DNA Ligase to create the lagging strand
1. Each strand of the DNA complex serves as a template
2. Both strands are replicated simultaneously • Discovered in 1960s by a Japanese molecular biologist Reiji and Tsuneko
3. Synthesis of the new DNA strand follows a 5’ to 3’ polarity or direction Okazaki
4. Substrates used are dATP, dGTP, dCTP, and dTTP
5. Replication is bidirectional
6. The Leading Strand is replicated continuously • Done by the 3’-exonuclease activity of DNA Polymerase in prokaryotes and
7. The Lagging Strand is replicated discontinuously eukaryotes
8. Okazaki Fragments are formed in the Lagging Strand • Reason for the high degree of fidelity of replication
9. Eukaryotic DNA has many origins of replication (Ori site) o Fidelity means faithfulness by which the DNA template is copied into
10. DNA Polymerase III cannot initiate so that it requires a DNA template and daughter DNAs. The accuracy of replication, so that in the end of
an RNA primer replication, the 2 daughter DNAs are actually replicas of the parent
DNA
• Mistakes can occasionally occur as when a DNA Polymerase inserts a wrong
• Done by DNA Polymerase I in prokaryotes and DNA Polymerase Delta in base
eukaryotes using their 5’-exonuclease activity • Most of the mistakes made during DNA Replication are promptly corrected
• Removes RNA primer nucleotide by nucleotide by DNA Polymerase 3’-exonuclease which proofreads the base that has just
been added
• Same enzyme replaces the ribonucleotide unit with complementary
deoxyribonucleotide acting on its polymerase mode • Uncorrected mistakes may sometimes lead to serious consequences such
o DNA Polymerase I – in prokaryotes as cancer
o DNA Polymerase delta – in eukaryotes • Repair mechanisms can correct the mistakes but in real cases, mistakes are
not corrected leading to mutations
• In other cases, Repair Enzymes are themselves mutated or defective

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 • tRNA is an adapter molecule with 2 active sites:
• Transcription is defined as the synthesis of RNA o Amino Acid Arm at its 3’ end that binds the Amino Acid
• Most of the regulations of Gene Expression takes place during o Anti-Codon Arm that binds to the mRNA
Transcription • It contains peculiar bases that are not found in mRNA and rRNA
• Genes encode Proteins, and Proteins dictate cell function • rRNA is needed in translation for proper alignment of Peptidyl Transferase
• Each step in the flow of information from DNA to RNA to Protein provides during peptide bond formation
the cell with the potential control of self-regulating its functions by
adjusting the amount and type of proteins it manufactures
• At any given time, the amount of a particular protein in a cell reflects the
balance between the Protein Synthetic and Degradative biochemical • A gene is a segment of DNA that determines a single character or
pathways phenotype
o On the Synthetic side of this balance, protein production starts at o Phenotype = observable characteristics of an organism
Transcription (From DNA to RNA) and continuous with Translation → Ex. Eye color, hair color, facial features, height, etc.
(RNA to Protein) o An organism’s phenotype is determined by its genotype, which is a set
o Control of these processes plays a critical role in determining what of genes that the organism carries, as well as the environmental
proteins are present in a cell and in what amount influences upon these genes
• In addition, the way in which a cell processes its RNA Transcript and Newly
Made Proteins also greatly influence protein level – called Post-
Transcriptional Modification • A gene is a segment of DNA that codes for:
o One enzyme
o One protein
A. Protein Coding RNA o One polypeptide
Messenger RNA (mRNA) contains the codons that specifies the sequence o One RNA
of amino acids in a protein • A gene is the entire DNA sequence required for the synthesis of functional
• RNA copy of the DNA found in the gene proteins or RNA molecule
• In addition to the coding regions, called exons, a gene includes
• The primary function of mRNA is to act as intermediary between
transcription control regions, and sometimes, introns, the non-coding
the genetic information in DNA and the amino acid sequence of region
proteins • In eukaryotes, genes are contained within a cell nucleus
• Each codon is recognized by specific tRNA to its anti-codon o The mitochondria in animals and chloroplast in plants also contains a
B. Non-Protein Coding RNAs small subset of genes distinct from the genes found in the nucleus
• Transfer RNA (tRNA) has the anticodon and the carrier of amino • In prokaryotes, genes are contained in a single chromosome that is free
acid floating in the cell’s cytoplasm
• Many bacteria also contain Plasmids, which are extrachromosomal genetic
o When a tRNA recognizes and binds to its corresponding
elements with a small number of genes
codon in the ribosome, the tRNA transfers the appropriate
amino acid to the end of the growing amino acid chain
• Ribosomal RNA (rRNA) becomes part of the ribosome
Cistron
o Responsible for reading the order of amino acids and linking
o Smallest unit of DNA which must be intact so that it can serve as
these amino acids together through the enzyme Peptidyl
transmitter of genetic information
Transferase
Muton
• Long Non-Coding RNA (LncRNA) is involved in chromatin
o Smallest unit of DNA whose alteration can give rise to a mutant form
remodeling of organism
• Small Nuclear RNA (snRNA) involved in removal of introns and Recon
splicing of exons o Smallest unit of DNA capable of recombination, presumably a series of
• Micro and Silencing RNA (mi/siRNA) modulates gene expression three nucleotide bases (triplet)
Recombination
o Formation of new combination of genes
The 3 RNAs that are directly involved in Protein Synthesis are: mRNA, tRNA, Introns (as in INtervene)
and rRNA o Intervening segments of DNA that do not code for the amino acid
tRNA and rRNA continues to decode the mRNA molecule until the entire sequence of the polypeptide (function is regulatory)
sequence is translated into a protein o In the nucleus, there are more introns than exons
Exons (as in EXpress)
• These are the primary RNA molecules involved o Coding segments of the gene
in Translation / Protein Synthesis o Coding sections of an RNA transcript that are translated into protein
• mRNA serves as a template for Protein Synthesis
o It is the direct carrier of information from During transcription of the gene, the RNA Transcript that is formed is called
nucleus to cytoplasmic ribosome, where Heterogenous Nuclear RNA (hnRNA) which contains the Introns and Exons.
translation takes place Introns are removed by RNA splicing as hnRNA matures, meaning that they
o Contains the codons that specify the amino are not expressed in the final mature mRNA product.
acid that will be incorporated to the
polypeptide or protein Most bacterial genes have no introns, whereas most genes of multicellular
o 2 Types of mRNA: organisms do.
→ Monocistronic and Polycistronic
→ Monocistronic mRNA is found in A gene consists of both coding sequence and regulatory sequences, while a
eukaryotes and it codes for only one cistron consist only of a coding sequence. Therefore, cistron resembles the
polypeptide coding sequence of a gene.
→ Polycistronic mRNA codes for 2 or
more polypeptides and is found in Cistron is the sequence of codons containing the complete information for
prokaryotes the synthesis of functional protein

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Chromosomes
o A structure in the nucleus containing the chromatin material made up
of:
→ 65% protein (histones)
→ 35% DNA
→ 5% RNA
o There are many genes in a single chromosome
→ E.g. E. coli = 3,000 to 5,000 genes
o Chromatin fibers resemble a string of beads
Nucleosome
o Repeating beadlike structures composed of:
→ Segment of DNA duplex (About 200 base pairs)
→ Eight histone molecules (2 each of H2A, H2B, H3 and H4)
Histones
Focus on the DNA on the upper part of the figure, you will notice the genes
o Found only in eukaryotes
located in the DNA. These are the lacI, lacZ, lacY, and lacA genes.
o Function to package and order DNA into structural units called
nucleosomes
lacZ gene is the structural gene for β-Galactosidase, which hydrolyzes
→ H1 → lysine-rich
lactose to galactose and glucose
→ H2A and H2B → large amount of lysine and arginine
lacY gene is the structural gene for permease, which transports lactose into
→ H3 and H4 → arginine rich the cell
lacA gene encodes a thiogalactoside transacetylase, which modify toxic
galactoside to facilitate their removal from the cell
• Structural Genes lacI gene encodes the repressor protein
o Genes that code for polypeptide and DNA
o Contain the codons that specify the amino acid The other components of the lac operon are the lac promoter and the lac
o Contain base sequence for primary structure of RNA operator, they are the regulatory regions in the lac operon.
• Non-Structural Genes
o Regulator genes that regulate the process of gene transcription Lactose is the inducer. When lactose and glucose are present, the cell
o Operator Genes metabolizes glucose first.
→ Serves as starting point for reading the genetic codes
→ Contains transcription starting site
o Regulator Genes
→ Synthesize repressor molecule that switches off the activity of the
structural gene

Operator Genes contain the code necessary to begin the process of

transcribing the DNA information present in the Structural Genes into
mRNA. Thus, Structural Genes are linked to an Operator Gene in a functional
unit called an Operon.

Regulatory Genes code for proteins that act like switches, turning other
A. Without Lactose or With Lactose and Glucose
genes on or off. These genes are essential to control cell function and
• In this illustration, there is no lactose (inducer).
without them, cells can grow out of control causing diseases like cancer in
• lac I gene is constitutively expressed to form 4 repressor subunits that
the body.
binds at the operator locus thus preventing the transcription of lac Z, Y,
and A genes by RNA polymerase
• lac I repressor is a negative regulator
• A constitutive gene is one that is regularly and constantly being
• A linear array of genes involved in a metabolic pathway transcribed and translated to a protein. It is a housekeeping gene.
E.g., lac operon and trp operon • In this particular case, it is lac I gene.
• Can be regulated by a single promoter region • The repressor subunits are being produced regularly regardless of
whether lactose is present or not.

• Smallest unit of genetic expression

• Codes for a subunit of a protein
• One Cistron, One Subunit Concept

• A single mRNA that encodes more than one separately translated protein
• Commonly found in prokaryotes

• Described by Francois Jacob and Jacques Monod in 1961

• They explained bacterial gene expression by studying lactose metabolism
in E. coli B. With lactose and no glucose
• Lac Operon describes how two adjacent genes involved in lactose • There is lactose (inducer) and no glucose
metabolism were coordinately regulated by a genetic element located at • Lactose molecules bind to repressor subunits thereby inactivating the
one end of the gene cluster repressors
• Repressors cannot bind to the operator site

Page 7 of 14
 • In the presence of cAMP and its binding protein (CAP, Catabolite Gene gene, initiation of RNA Synthesis, elongation of RNA Transcript, and
Activator Protein, cAMP Regulator Protein [CRP]), RNA polymerase can termination of RNA Synthesis.
now transcribe the structural genes Z, Y and A into their corresponding RNAP Holoenzyme is a hexamer, made up of 6 subunits (2 alpha, 2 beta, 1
enzymes omega, 1 sigma)

The interaction between the core enzyme and the sigma factor results in an
RNAP holoenzyme that can detect specific promoter sequences in the DNA
1. Takes place in the nucleus template and activate transcription in various conditions.
2. Requires DNA template
3. Read the template in 3’ to 5’ direction Bacteria contain a single type of RNAP, while eukaryotes contain 3 distinct
4. Incoming nucleotide units are added to the 3’ end of the DNA or RNA types (RNAP I, II, III), which are differentiated by their sensitivity to a peptide
Primer toxin from the mushroom Amanita phalloides.
5. New strands are synthesized in 5’ to 3’ direction
6. Follows the general steps of initiation, elongation, and termination
7. Adheres to Watson-Crick base pairing rule

1. In Replication, the entire DNA template is copied while in

Transcription, only segments of DNA are copied
→ The segment refers to gene in the DNA that is copied into an
RNA
2. Ribonucleotides are used in RNA Synthesis
3. Uracil replaces Thymine as complementary base for Adenine in RNA
4. An RNA Primer is involved in replication but not in transcription
because what you’re synthesizing in transcription is the RNA itself RNAP locally opens the double stranded DNA, producing a transcription
5. There’s no efficient proofreading mechanism during transcription bubble so that one strand of the exposed nucleotides can be used as a
6. Replication: DNA Polymerase; Transcription: RNA Polymerase template for the synthesis of RNA. The illustration shows the transcription
bubble and the RNAP holoenzyme.
Replication Transcription
Cellular Localization Nucleus Nucleus During transcription, the DNA of the gene must separate into coding strand
Need for DNA Template Required Required and template strand. The black colored strand above is the template strand.
Parts of DNA Copied Entire DNA Segments of DNA It is the one being copied (transcribed) in order to form the RNA transcript.
Number of Strands 2 1 Only one of the DNA strands is being copied.
Copied
Need for RNA Primer Yes No
Major Enzyme DNA Pol III RNA Pol 1. Template Binding (Recognition)
Direction of Elongation 5’ to 3’ 5’ to 3’ 2. Chain Initiation
NTPs Required dATP, dGTP, dCTP, ATP, GTP, CTP, UTP 3. Chain Elongation
dTTP 4. Termination and Release
5. Post-Transcriptional Modification of RNA Transcript

• Principal enzyme involved in Transcription

• Involved in all steps of Transcription – recognition, initiation, elongation, • Recognition of specific binding site (promoter site) in the DNA template
and termination • Characteristics of the Promoter Site:
• RNAP holoenzymes consists of 2 alpha, 2 beta, 1 omega and 1 sigma o Pyrimidine-Rich Region
subunits → Pyrimidines found in DNA: Thymine and Cytosine
o Core Enzyme – consists of 2 alpha, 2 beta, and 1 omega subunits o Targe of Regulatory Effectors
o Sigma Subunit (Sigma Factor) – regulatory subunit; helps the core → Effectors are called Modulators – they regulate gene
enzyme to attach more tightly to specific site in the promoter region transcription
• Types of Mammalian RNA Polymerases: → Positive Modulator – stimulates gene transcription
o RNAP I (A) → Negative Modulator – inhibit gene transcription
→ Insensitive to Amanitin o Sigma Factor enhances binding of RNA Polymerase
→ Synthesize rRNA
→ Amanitin – toxic principle found in poisonous mushroom Promoters are DNA sequences whose purpose is not to encode information
(Amanita phalloides) about the organism itself but rather they serve as a kind of “on switch” to
o RNAP II (B) initiate the biological process of transcription. A promoter is a region of DNA
→ Sensitive to low concentration of Amanitin where transcription of a gene is initiated. The enzyme RNAP, which
→ Synthesize hnRNA (mRNA), IncRNA, miRNA, SnRNA performs the transcription process, binds the promoter sequence.
o RNAP III (C)
→ Sensitive to high concentration of Amanitin Regulatory Effectors are molecules that act as ligands that can increase gene
→ Synthesize tRNA expression. A ligand is a substance that forms a complex with a biomolecule
to serve a biological purpose. In DNA-ligand binding studies, the ligand can
RNA Polymerase is an enzyme that is responsible for copying a DNA be a small molecule, iron, or protein, which binds to the DNA double helix.
sequence into an RNA sequence during the process of Transcription.
It is DNA-dependent because RNA Polymerase requires a DNA template to
initiate transcription.
One advantage of RNA Polymerase over DNA Polymerase is that it is involved
in all the steps of transcription. From recognition of the Promoter Site of the

Page 8 of 14
 → Designated as -10; therefore, start site is located 10 sequences
upstream from TATAAT Box
o TGTTGACA
→ Frequency signal
→ Tells the number of times sequences should be transcribed

• The figure illustrates the 2 strands of the DNA duplex.

• In transcription, only 1 of the 2 strands is copied into an RNA molecule
→ Template Strand
• Template Strand is the one shaded in the illustration.
o In Genes A, B, and D, the template strand is the lower strand
o In Gene C, the template strand is the upper strand
o This means that there is no fixed strand, it can be any of the two
strands (upper or lower) Eukaryotic Promoter Site
• The Template Strand is the DNA strand that is transcribed into an RNA. It is almost similar with the prokaryotic gene. +1 is where the TSS is. The
It is also called Non-Coding or Antisense Strand. TATA box (Hogness box) provides the “where” signal and confers “fidelity”
• The Coding (Sense) Strand is the untranscribed DNA strand of initiation (accuracy of initiation of transcription). It tells that 32 bases in
o Thus, a given strand of a double stranded DNA will serves as the front is the TSS. The CAAT box determines the frequency of transcription
template strand for some genes (A, B, D) and the coding strand of initiation.
other genes (C)
The TATA Box is usually located 25 to 35 base pair upstream from TSS in
• Note that the template strand is always read by RNAP in the 3’ to 5’
mammalian genes. This signals RNAP on where the TSS is. It confers fidelity
direction, as shown in the arrow head in the illustration.
or accuracy of initiation of transcription.
o In Genes A, B, and D, the direction of the arrow runs from 3’ to 5’
direction. The CAAT Box and GC Boxes determines the frequency of transcription
o This is how RNA Polymerase reads the base sequence of the DNA initiation or how frequent the gene will be transcribed. The frequency is
Template (3’ to 5’) increased by the presence of these cis acting elements.
o The RNA Transcript is being synthesized anti-parallel, from 5’ to 3’
The Latin prefix means “on the site” or on the same molecule of DNA as the
direction
genes to be transcribed.
o Same in Gene C, the template strand runs on a 3’ to 5’ direction
and its RNA Transcript is synthesized from 5’ to 3’ direction
Cis regulatory elements are often binding sites for 1 or more trans acting
protein factors. Trans regulatory elements are genes which may modify or
What is the importance of Coding Strand?
regulate the expression of distant genes. More specifically, trans regulatory
It is where you find the regulatory sequences in the promoter region of the
elements are DNA sequences that encode trans acting factors. These are
gene
often proteins, such as transcription factors.

To summarize, cis regulatory elements are present to the same molecule of

DNA as the gene they regulate, whereas trans regulatory elements can
regulate genes distant from the genes from which they were transcribed.

GC boxes and CAAT boxes – cis acting elements

CTF, TFIID, Sp1 – trans acting protein factors

Prokaryotic Promoter Site Coding Strand contains the following Regulatory Genes: Eukaryotes
The Transcription Start Site (TSS) is indicated as +1. All nucleotides before • Hogness Box – TATA Box
and upstream of the start site are given negative numbers and they are o Found in Eukaryotes
referred to a 5’ Flanking Sequence. Nucleotide sequences after and o Provides the “where” signal in eukaryotes
downstream from the start site, the 3’ Flanking Sequence, is the transcribed o Confer “fidelity” of initiation
region of the gene and is given positive numbers. Take note that the • CAAT Box
promoter region is found upstream of the TSS. o Determines the frequency of transcription initiation

The DNA regulatory sequence elements, such as the -35 and -10 TATAAT
box are described in a 5’ to 3’ direction as they are located in the Coding • Alignment of first 2 nucleotide triphosphates (NTPs) to their
Strand of the DNA. The TATAAT Box (Pribnow Box) signals the RNAP where complementary bases on the DNA template
the TSS is. o 1st NTP (forms the 5’ end): ATP or GTP
The letters in the box are deoxyribonucleotide units which are base o 2nd NTP: UTP or CTP
sequence in the coding template that serve as regulatory signals. The • First NTP + RNA Polymerase = Initiation Complex
TATAAT box (Pribnow box) confers fidelity of initiation of the transcription
start site. It is the one that will tell the RNA polymerase that the TSS is 10 To begin transcribing a gene, RNAP binds to the DNA of the gene at the
bases away from it (TATAAT box). The TGTTGACA is a regulatory signal that region called the promoter. Basically, the promoter tells the polymerase
tells the RNA polymerase the frequency of initiation of the gene (how many where to sit down on the DNA and begin transcribing. Before transcription
times it will transcribe the gene). can take place, the DNA double helix must first unwind near the gene that
is being transcribed. The region of opened up DNA is called the Transcription
Bubble, separating the template strand from the coding strand. This way,
Coding Strand contains the following Regulatory Genes: Prokaryotes
RNAP can now read the bases in the template DNA strands. The TSS
o Pribnow Box – TATAAT Box
correspond to the 5’-end of the RNA Transcript being formed. There will be
→ Found in Prokaryotes
alignment of the first 2 Nucleotide Triphosphates (NTPs) to their
→ Confers the “where” signal in prokaryotes
complementary bases on the DNA template. Usually, but not always, it is a
→ Denotes Transcription Start Site for RNA Polymerase
Purine NTP, either ATP or GTP. The 2nd NTP to base pair with the base on the
DNA template is either UTP or CTP.

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During the condensation of the 2 NTPs, a pyrophosphate will be released so
that only mononucleotide units will be bonded together by RNAP with a
phosphodiester bond. The pyrophosphate will be acted upon by
pyrophosphatase, releasing 2 inorganic phosphates and energy, thereby
providing irreversibility to the overall synthetic reactions.

1. Sigma factor dissociates from one core RNA Polymerase

2. DNA template is read from 3’ to 5’ direction
3. Direct elongation is from 5’ to 3’ direction Transcription Termination Signal
4. New nucleotide units are added to 3’ end of growing RNA polymer
Rho-Independent Termination depends on specific sequences in the DNA
Chain elongation is the stage when the RNA strand gets longer due to the template strand. As the RNAP approaches the end of the gene being
addition of new nucleotides. During elongation, the RNAP walks along the transcribed, it hits a region rich in Cytosine and Guanine nucleotides. The
DNA template in the 3’ to 5’ direction. For each nucleotide in the template, RNA transcribed from this region folds back on itself and the
RNAP adds a matching or complimentary RNA nucleotide to the 3’-end of complimentary cytosine and guanine nucleotides bind together. The result
the growing RNA strand. The RNA Transcript is being elongated in a 5’ to 3’ is a stable hairpin loop that causes the polymerase to stall.
direction, antiparallel to the template strand.
In eukaryotic cells, termination is less well understood.
RNAP has an intrinsic unwindase that opens the DNA helix, similar to that of
helicase. The unwinding causes a disruption in the nucleosomal structure of
the DNA. Topoisomerase both precedes and follows the progressing RNAP
to prevent the formation of superhelical tensions or supercoils that will 1. Addition of nucleotides to 3’ and 5’ terminal
increase the energy required to unwind the DNA template. a. Methylated G Nucleotide (called CAP) is added at 5’ end
b. Long stretch of Poly-A Nucleotides is added to 3’ end
RNAP II is responsible for transcribing DNA into mRNA. Unlike DNA 2. Removal of introns and splicing of exons
Polymerase, it does not have a 3’ exonuclease proofreading activity. Errors
in transcription can cause deleterious effect upon repeated translation of DNA Transcription occurs in the cell’s nucleus. In prokaryotes, the RNA that
erroneous mRNA into protein. Transcription Infidelity may result in aging is synthesized during transcription is ready for translation into a protein.
and human diseases such as cancer. Eukaryotic RNA, however, is not immediately ready for translation, it has to
undergo Post-Transcriptional Modification.
During transcription, Pol II can detect the disincorporated RNA and
backtrack to correct errors to ensure that each mRNA created will match There are 3 processes that make up this Post-Transcriptional Modification.
with the template DNA. However, it remains largely a mystery how Pol II o 5’ capping
controls the fidelity of gene transcription. o Addition of the Poly-A Tail
o Splicing

1. DNA template contains Stop Signals The 5’ Capping Reaction replaces the triphosphate group at the 5’ end of
o GC-rich region followed by AT-rich region the RNA chain with a special nucleotide that is referred to as the 5’ CAP.
2. Two Major Termination Strategies found in bacteria o It is made up of a methylated Guanine nucleotide
o Rho-Dependent Termination o It is thought to help with mRNA recognition by the ribosome during
→ Rho protein-dependent termination – the protein is an translation
ATPase with helicase activity
→ Helicase separates the RNA-DNA hybrid helix causing the A modification also takes place at the opposite end of the RNA Transcript.
release of RNA transcript To the 3’ end of the RNA chain, there are 3 to 500 adenines are added in
→ The RNA contains a binding site for a protein called Rho what is called a Poly-A Tail.
Factor o Formation of this Poly-A Tail is quite expensive because it comes
→ Rho Factor binds to the sequence and starts climbing up the from hydrolysis of ATP
transcript towards RNAP, when it catches up with the
polymerase at the Transcription Bubble, Rho pulls the RNA Removal of Introns, the non-coding segment, and splicing or joining
Transcript and the DNA template strand apart, releasing the together of Exons, the coding segment, takes place during the maturation
RNA molecule and ending transcription of the heterogenous nuclear RNA (hnRNA) to a functional mature mRNA.
o Rho-Independent Termination This process involves spliceosomes, a multicomponent complex made up of
→ Rho protein-independent termination allows the RNA the RNA Transcript, small nuclear RNA (snRNA), and a number of some other
transcript to fold back on itself forming a “hairpin” loop since proteins. Collectively, they form a small ribonucleotide protein termed
the nascent RNA has a nucleotide sequence that is self- snRNP Complex.
complementary
→ Causes the RNAP to pause and helps Rho to catch up

Rho factor is a prokaryotic protein involved in termination of

transcription

• This figure shows the Post-Transcriptional Modification of mRNA showing

the 7-methylguanosine cap and Poly-A Tail

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 • When a DNA binding protein binds to an enhancer, the shape of the DNA
A. Inhibition that binds to the DNA template changes.
1. Actinomycin D – antibiotic • An enhancer is a DNA sequence that promotes gene transcription.
o Shown to have the ability to inhibit transcription by binding DNA at the • In genetics, a silencer is a DNA sequence capable of binding transcription
Transcription Initiation Complex and preventing elongation of RNA regulation factors called Repressors.
chain by RNAP • When a repressor protein binds to the silencer region of DNA, RNAP is
o Also called Dactinomycin prevented from transcribing the DNA sequence into RNA.
o A well-known antibiotic that exhibits high antibacterial and anti-tumor • With transcription blocked, the translation of RNA into proteins is
activity impossible.
2. Aflatoxin – derived from fungus Aspergillus flavus and Aspergillus parasiticus, • Silencers prevents genes from being expressed into proteins.
associated with hepatoma
o Produced by certain fungi that are found in corn, peanuts, cotton seed,
and tree nuts
o Aspergillus flavus and Aspergillus parasiticus which are abundant in
warm and humid regions in the world
o Poisonous carcinogens and mutagens

B. Inhibitors that bind to RNA Polymerase

1. Rifampicin – an anti-TB drug
o Antibiotic used to treat Tuberculosis and Leprosy
o It prevents RNA Synthesis by physically blocking elongation and thus, • Bind with high affinity to the correct region of the DNA
preventing synthesis of post-bacterial proteins • 3 unique motifs that enhance binding of the proteins to DNA
2. Amanitin – toxic principle found in poisonous mushroom Amanita phalloides o Helix-turn-helix
(death cap) o Zinc finger
o Cyclic octapeptide which inhibits RNAP II, thus interfering with Protein o Leucine zipper
Synthesis • The protein provides a ligand-binding site for modulators or effectors

• Increase the number of genes available for transcription results in increase

o E.g., B-Globin gene cluster is in “active” chromatin (euchromatin) in in the number of RNA and proteins
the reticulocyte but in “inactive” chromatin (heterochromatin) in • Generated by the process of repeated initiations during DNA synthesis
muscle cells thereby providing multiple sites for gene transcription
• This is the basis of development of drug resistance to methotrexate by
If the genome is the same in all somatic cells within an organism, how do increasing the number of dihydrofolate reductase
the cells become different from one another?
If every cell in the body contains the genes for hemoglobin and insulin
proteins, how are the hemoglobin proteins made only in RBCs and how • Process involved in the synthesis of different antibodies by lymphocytes
are insulin proteins made only in certain pancreatic cells and neither are • It allows the generation of 109-1011 different immunoglobulins from a
made in the kidney or nervous system? single gene
• The variable region of the antibody is the result of somatic recombination
Answer: The answer is through the process of Differential Gene of segments within both the light and the heavy-chain genes
Expression, which is the activation of different genes within a cell that
define its purpose. Each cell expresses only those genes which it needs.
However, the extra genes are not destroyed but continue to be stored • Mobile segments of DNA that moves in a random manner from one site to
within the nucleus of the cell. another on the same or different chromosome
• Mediated by transposase, an enzyme that cuts out and inserts the Tn at a
The 2 types of chromatin, heterochromatin and euchromatin, are new site and is copied
functionally and structurally distinct regions of the genome. • Replicative transposons – may involve an RNA intermediate and called
Heterochromatin is densely packed and inaccessible to transcription retrotransposons
factors so it is rendered transcriptionally silent. Euchromatin is less o May cause disease such as Hemophilia A and Duchenne muscular
dense, more accessible, and therefore transcriptionally active. dystrophy
• Exchange of plasmids that contain transposons carrying antibiotic
resistance to the recipient bacteria
o E.g., Histone acetylation by acetylase
→ Acetylation of lysine residues in amino terminal tails
→ Reduced positive charge of these tails • Promoter region of the gene contains CpG islands (CG-rich region)
• Transcriptionally active genes are less methylated
In eukaryotes, DNA is tightly wound into a complex called Chromatin. • This suggests that DNA hypermethylation silences gene expression
Due to the process of Chromatin Remodeling, this complex can be • Methylation is catalyzed by methyltransferases using S-
opened so that specific genes are expressed. adenosylmethionine (SAM) as the active methyl group donor
An example of this is acetylation of lysine residues present in histones.
Histones are positively charged and DNA is negatively charged.
Acetylation results in decreased binding of histones to DNA, thus
opening up the nucleosomal structure. • Protein Synthesis is accomplished through a process called Translation
• After DNA is transcribed into an mRNA molecule during transcription, the
mRNA must be translated to produce a protein
• Enhancers are DNA regulatory elements that activate and increase • In translation, mRNA along with tRNA and ribosomes work together to
transcription of a gene produce proteins
• Enhancer Regions are binding sequences or sites for transcription factors

Page 11 of 14
• Contains 5 stages:
1. Activation of amino acid
2. Initiation of the polypeptide chain
3. Elongation of the polypeptide chain
4. Termination of the polypeptide chain
5. Post-translational processing

Why is it important to study this?

Without transcription and translation, our body would have no possible way
to make proteins or functions. Proteins allow our body to do everything.
o Muscle proteins allow skeletal muscles to strengthen and grow.
o Antibodies protect the body from germs.
o Some proteins support body structures.
o Others help with body movements. • 70s ribosome is inactive
o There are thousands of functions for different proteins. • Must first dissociate into their 30s and 50s subunits
• Initiating f-met-tRNA can bind only to P site
o An exception to the rule because other aminoacyl-tRNA complex binds
• Involves the formation of aminoacyl-tRNA complex first to A site
• Catalyzed by aminoacyl-tRNA synthetase which is highly specific • Translation of codon of mRNA is 5’ to 3’ direction
• N-terminal amino acid is 1st part of protein to be formed
Amino Acid + tRNA + ATP-Mg
Synthetase The mRNA serves as a template in translation. The first part of the ribosome
to bind to the mRNA is the smaller 30s subunit, followed by the large 50s
Aminoacyl-tRNA + AMP + PPi subunit, to form a complete ribosome.

During Amino Acid activation, the amino acids are attached to their The 50s subunit contains the P Site (Peptidyl Site). The initiating formyl-
corresponding tRNA. The coupling reactions are catalyzed by a group of methionyl tRNA complex binds to this P Site.
enzymes called Aminoacyl-tRNA synthetases. Each of the 20 amino acids are It also has an A Site (Aminoacyl Site) to which the incoming aminoacyl-tRNA
recognized its specific aminoacyl-tRNA synthetase. complex will bind.

The base sequence of the codon in mRNA is read by peptidyl transferase in

a 5’ to 3’ direction. The first part of the protein to be formed is the N
Terminal.

1. Binding of mRNA to the smaller subunit (30s) of ribosome

2. Formation of initiation complex
o Aminoacyl-tRNA base pairs with corresponding codon on mRNA
3. Initiating codon: AUG
Initiating amino acid: N-formylmethionine (prokaryotes)
Methionine (eukaryotes)
4. Other requirements: GTP, Mg, Initiation Factors - IF1, IF2, IF3
5. Attachment of large ribosomal unit (50s)
6. Inhibited by Streptomycin

1. An adapter molecule
o Amino Acid Arm – binds the amino acid • Starts when initiating formyl-methionly-tRNA complex is bound to the P
o Anticodon arm – recognizes the codon in mRNA Site
o Enzyme recognition site – peptidyl transferase
o Ribosome attachment site – large ribosomal subunit 1. Binding of Incoming Aminoacyl-tRNA Complex
tRNA plays a very important role in protein synthesis. As an adapter a. Attaches to the A Site
molecule, it has many parts. b. Requires Elongation Factors (EF-T and EF-G) and GTP
o Amino Acid Arm at the 3’ end that binds the amino acid and → Energy is provided by hydrolysis of GTP
carries it to the ribosome c. Blocked by Tetracycline
o Anticodon Arm is complimentary to the codon present in 2. Peptide Bond Formation
mRNA. a. Catalyzed by Peptidyl Transferase (ribozyme)
o Its enzyme recognition site allows it to interact with b. Enzyme is inhibited by Chloramphenicol
peptidyl transferase in peptide bond formation. c. Product is Dipeptidyl-tRNA bound to A Site
o It delivers the amino acid to the A site of the large → In this condensation step, the initiating amino acid,
ribosomal subunit which is Methionine, will disassociate from its tRNA in
the P Site and forms a peptide bond with the 2nd amino
2. High content of peculiar bases acid
o E.g., inosine, dihydrouridine, pseudouridine and methylated bases 3. Translocation
3. Cloverleaf appearance (twisted letter “L”) a. Ribosome moves to the next codon of mRNA
b. Peptidyl-tRNA shifted from A Site to P Site
c. Requires EF-G and GTP
d. Inhibited by Erythromycin

• Occurs continuously until all the codons in the mRNA have been translated
into the protein molecule

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 Inhibitor Action
Coumarin (-) DNA Gyrase • 20 different amino acids compose a protein molecule
Quinolone (-) DNA Gyrase • There are 4 code letters in DNA – A, T, G, and C
Rifampicin (-) RNA Polymerase by binding to Beta Chain • 3 nucleotide residues of DNA are required to code for each amino acid
Chloramphenicol Binds to 50s o If only 2 letters/nucleotides, 42 = 16 codons (insufficient)
(-) Peptide Bond Formation o If 3 letters/nucleotides, 43 = 64 codons (more than enough to cover the
Erythromycin (-) Translocation 20 different amino acids)
Fusidic Acid (-) Translocation
Puromycin Amino Acid-tRNA analogue causing premature chain
termination • The genetic code is universal
Tetracycline Binds to 30s subunit o Amino acid code words are identical in all species
(-) Amino Acid-tRNA binding to A site • The genetic code is commaless
Cyclohexamide (-) Peptidyl Transferase on eukaryotic 60s o No punctuation or signal is required to indicate the end of one codon
Diphtheria Toxin (-) Eukaryotic EF-2 by ADP Ribosylation and the beginning of the next
Streptomycin Binds to 30s subunit o This is the basis for Frameshift Mutation
Causes mRNA misreading → If you add or delete a nucleotide, the reading frame of the
Kanamycin Binds to 70s subunit sequence of bases in the genes will change and because of that,
Causes mRNA misreading you will incorporate a different amino acid because of the change
in sequence of codons
• The genetic code is non-overlapping
1. Presence of termination signal in RNA o Each group of 3 bases (codon) specifies only 1 amino acid
2. UAG, UGA, UAA • The genetic code is degenerate
o Stop Codon/Nonsense Triplets o Different amino acids have different number of codons
o Do not code for any amino acids → There are 61 codons in total, some amino acids may have more
3. Requires Releasing Factors (RF 1, RF 2, RF 3) than 1 codon
When the Releasing Factors occupies the A Site, there will be dissociation → Leu, Ser = 6 codons each
of the complex. → His, Tyr = 2 codons
o Hydrolytic cleavage of polypeptide → Trp, Met = 1 codon
o Release of empty tRNA from P site o Degeneracy only involves the third base in the codon for most amino
o Dissociation of 70s ribosomal unit into 50s and 30s subunits acids
→ 3rd base is less specific than the first 2
This happens when the ribosome encounters a stop codon in the mRNA. → Ex. Alanine is coded by GCU, GCC, GCA
There are 3 Stop Codons: UAG, UAA, UGA. There is no anticodon that → 3rd Base is called the Wobble Base
matches these three nonsense triplets. They do not code for any amino acid.
→ Wobble Base allows some tRNA to recognize more than 1 codon
o When 2 different amino acids have code words in which the first 2
UAG = “amber” bases are identical, the 3rd base of one is filled only with purines and
o First stop codon to be discovered the 3rd base of the other one is filled only with pyrimidines
o Charles Steinberg and Richard Epstein, the discoverers, named UAG → Histidine: CAU
“amber” after the surname of their friend Harris Bernstein, which CAC = Pyrimidine
means amber → Glutamine: CAA
UAA = “ochre” CAG = Purines
UGA = “opal”
o For the other stop codons, they just named these after colors to be
consistent with the “amber” motif

1. N-terminal and C-terminal modification

o
E.g., removal of formyl group from f-Met by deformylase
2. Loss of signaling sequences
o These are extra amino acid residues found in the N-terminal end of the
polypeptide
o This “leader” peptide directs the protein to its ultimate destination in
the cells (which is the cisternae or lumen of the Rough ER)
o They are removed by specific peptidases

The newly synthesized protein will undergo Post-Translational Processing or

Modification. Some proteins, especially those that are destined to be
exported out of the organ where they are synthesized, contains extra amino
acid residues in their N Terminal end, this is called the Leader Peptide or the
Signal Peptide.

Not all protein that is synthesized needs a leader peptide.

What proteins are synthesized with a leader peptide? Proteins that are
destined to be exported out of the cell. Example would be the Alpha-1-
Antitrypsin which is synthesized in the liver which is extruded out of the
bloodstream going to the lungs to inhibit the enzyme elastase (a degradative
enzyme that destroys elastin in the alveolar wall). If elastase is not inhibited,
COPD can occur such as emphysema

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 • After translation of mRNA, there is now a garbled sequence of amino acids
• A permanent alteration in the DNA sequence in the new protein which is very different from the sequence in the original
• The sequence differs from what is found in most people protein
• Ranges in size
• Can affect anywhere from a single DNA base pair to a large segment of a
chromosome (multiple genes)

• Inherited from a parent

• Present throughout the person’s life in virtually every cell in the body
• Present in the parent’s egg or sperm cells (germ cells)

• Occur at some time during a person’s life

• Present only in certain cells
• Can be caused by environmental factors (e.g., UV radiation) or as a mistake
during DNA replication
• Variation in the DNA sequence that occurs in a population with a frequency
• Acquired mutation in somatic cells cannot be passed on to the next
of 1% or higher
generation
o DNA sequence variation that is common in the population
o No single allele is regarded as the standard sequence
• Genetic mutations that are described as de novo can be either
• Higher incidence in the population suggests that polymorphism is
hereditary or somatic
naturally-occurring
• In some cases, mutation occurs in a person’s germ cell but is not
present in any of the person’s other cells
• In other cases, the mutation occurs in the fertilized egg shortly after the
• Any rare change in the nucleotide usually but not always with a disease-
egg and sperm unite
causing attribute
• As the fertilized egg divides, each resulting cell in the growing embryo
• Any change in a DNA sequence away from normal
will have the mutation
• Implies that there is a normal allele that is prevalent in the population
• De novo mutations may explain genetic disorders in which an affected
o Mutations changes this to a rare and abnormal variant
child has a mutation in every cell in the body but the parents do not and
there is no family history of the disorder

• Change in a single base

• Transition
o Purine to purine
o Pyrimidine to pyrimidine
• Transversion
o Purine to pyrimidine and vice versa

• No detectable effect because of the degeneracy of the genetic code

• Missense effect (missense mutation)
• Different amino acids are incorporated
o Could be an acceptable, partially acceptable, or unacceptable missense
based on resulting functionality of the protein product
• Nonsense codon appears resulting in premature termination of the protein

• Altered sequence of mRNA

• Either a deletion or insertion frameshift mutation

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