BIO20 Introduction to Biomimetic Engineering and Component Design
Central Dogma of Molecular Genetics
INFORMATION PATHWAYS
Gene is the smallest unit of inheritance. It is a
segment of DNA that encodes the information
required to produce a functional biological product.
The units of information are copied or converted
into functional products through three major
processes: The Central Dogma of Molecular
Genetics
Replication – copying of parental DNA to form
daughter DNA molecules having identical
nucleotide sequences.
Transcription – process by which parts of the coded
genetic message in DNA are copied precisely in the
form of RNA.
Translation – in which genetic message coded in
mRNA is translated on the ribosome into a protein
with a specific sequence of amino acids.
DNA’s are packaged into chromosomes.
A chromosome contains thousands of individual
genes. The sum of all genes on all the different
chromosomes of a cell is referred to as genome.
Viral DNA molecules are small.
DNAs found in some viruses are single stranded.
The genomes of viruses are either made up of DNA
or RNA.
Bacteria contain much more DNA than viral DNA.
Bacteriophage DNA is 17.5 µm long while that of an
E. coli is 1.7 mm long. Eukaryotic cells contain
more DNA than Prokaryotes.
The total length of DNA in a single human cell is
about 2 meters.
Approximately 104 cells of adult, DNA would be
14 11
about 2 x 10 m or 2 x 10 km.
Genes are segments of DNA that codes for
polypeptide chains and RNA’s.
How many genes in a single chromosome?
The average gene of an E. coli is 1,050 base pairs
long.
Genes encode for:
a) polypeptide or RNAs (structural genes)
b) regulatory genes
Eukaryotic chromosomes are very complex.
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Test made in mouse DNA:
10% of the DNA consists of short lengths of less
than 10 base pairs that are repeated millions of
times per cell. highly repetitive
20% of the DNA consists of about hundreds of base
pairs repeated at least a thousand times per cell.
moderately repetitive
70% of the DNA are repeated only few times.
(unique sequence of base pairs)
Satellite DNA – most highly repeated sequence.
Junk DNA – some repetitive DNA.
Centromere – attachment site for proteins that
links chromosomes.
Many eukaryotic genes
contain intervening
non-transcribed sequence.
Nucleotide sequences of Eukaryotic genes contain
one or more intervening segments of DNA that do
not code for the amino acid sequences of the
polypeptide product.
Such non-translated DNA segments in genes are
called intervening sequence or introns and the
coding sequences are called exons.
Example:
gene coding for single polypeptide chain of the
avian egg protein ovalbumin contains 7 introns and
8 exons. Introns make up 85% of DNA of this gene.
The Human Genome Project:
Genome is the complete set of genetic information
of an organism
The human genome project:
- 2001 Draft of Human Genome Sequence
- 2003 Finished Human Genome
- (50 years after DNA structure solved)
- Costed $3 billion
The total length of the human genome is over 3
billion base pairs.
Significance:
to study gene expression in a specific tissue, organ
or tumor; to study human variation; to study how
humans relate to other organisms; to find
correlations how genome information relates to
development of cancer, susceptibility to certain
diseases and drug metabolism
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 BIO20 Introduction to Biomimetic Engineering and Component Design
DNA REPLICATION
DNA replication is an essential aspect of cellular
and viral reproduction. Replication of a double-
stranded DNA results in two double-stranded DNAs
as products.
Some important general points about DNA
replication are as follows:
Replication is exceedingly accurate--far more
accurate than any other enzyme-catalyzed process.
Replication is rapid.
The mechanism of replication is semi-conservative-
-each newly made strand is copied from one of the
parental strands and the products of replication are
two molecules, each containing one parental strand
and one newly synthesized strand.
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Central Dogma of Molecular Genetics
Replication is orderly and sequential--it begins at a
fixed point called an origin (or ori) and closely
follows parental duplex unwinding. Replication can
be broken down into three processes--initiation,
elongation, and termination.
DNA replication uses deoxynucleoside-5'-
triphosphates (dNTPs) to build the DNA chains.
DNA replication is discontinuous--synthesis of one
strand (called the lagging strand) lags behind the
other (called the leading strand) and occurs in
pieces called Okazaki fragments. Replication of
the leading strand is continuous.
DNA chain growth (replication) proceeds only in the
5' to 3' direction. DNA polymerase catalyzes the
chemical reaction of DNA synthesis.
DNA replication is should be accurate.
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 BIO20 Introduction to Biomimetic Engineering and Component Design
Rate of DNA Replication
Multiple proteins are required for DNA replication at
a replication fork such as DNA helicases, DNA
polymerases, topoisomerases, and DNA ligase.
Some of these are multisubunit protein complexes.
There are three major steps in DNA Replication:
INITIATION
Helicases actively denature DNA and actively
unwind duplex DNA strands. It provides this
function by catalyzing the ATP-dependent
unwinding of double-strand DNA.
Topoisomerases are enzymes with a "swivel"
mechanism that can relieve torsional stress.
Bidirectional replication of the circular E. coli
chromosome unwinds about 100,000 base pairs per
minute. At 10 bp per turn, this is 10,000 turns of
DNA per minute or over 160 per second. If there
were nothing to relieve this stress, DNA would be a
tangled mess in seconds. Relief of torsional stress
is essential for DNA replication to occur.
ELONGATION
Replication of DNA occurs at a molecular junction
that is usually drawn schematically as a fork and is
hence called a replication fork. The leading strand
and lagging strand replication occur on opposite
strands at the same replication fork and that
replication proceeds for both strands in the 5' to 3'
direction.
Primase - DNA polymerases cannot initiate the
synthesis of new DNA chains, but can only extend
chains from preexisting 3' hydroxyl termini. Initiation
of new DNA strands in E. coli begins with RNA
fragments. Primase is the enzyme responsible for
catalyzing synthesis of these primers.
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Central Dogma of Molecular Genetics
DNA polymerases – synthesizes the new DNA
strand in the 5’ to 3’ direction anti-parallel to the
DNA template strand
Leading strand - the strand of DNA at a replication
fork that replicates continuously.
Lagging Strand - the strand of DNA at a replication
fork that replicates in pieces (Okazaki fragments).
Okazaki fragment - short discontinuous stretches of
DNA arising from replication on the lagging strand.
PROOF-READING IN REPLICATION
DNA replication is, by far, the most accurate of
known enzyme-catalyzed processes. The error rate
-9
per base pair per round of replication is about 10
-10
to 10 . The accuracy cannot be explained,
however, by the different binding energies between
correct and incorrect base pairs (this amounts to
only a 100 to 1000 fold difference between correct
and incorrect pairing).
Two cellular systems aid the fidelity of replication.
These include the following:
1. The 3'-5' exonuclease activity of DNA
polymerases acts as a "proofreading" system.
Incorrectly incorporated nucleotides are not readily
extended by the polymerase very efficiently,
allowing them to be melted away to the 3'-5'
exonuclease site of the polymerase for removal.
2. The mismatch repair system makes an additional
contribution to accuracy of about 100-fold. It works
by scanning the newly replicated DNA, excising any
residues that are not properly base-paired and
replacing them with the correct nucleotides.
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 BIO20 Introduction to Biomimetic Engineering and Component Design
TERMINATION
DNA ligase - Covalently closes nicks in double-
stranded DNA. The nick must contain 3' hydroxyl
and 5' phosphoryl termini and the nucleotides being
linked must be adjacent in a duplex structure and
properly base-paired. DNA ligase functions to seal
Okazaki fragments in the lagging strand of DNA
replication.
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Central Dogma of Molecular Genetics
TRANSCRIPTION
- process of RNA Synthesis
- copying the 5’ – 3’ DNA in the form 5’ – 3’ RNA
-synthesis of 5’ to 3’ RNA antiparallel to DNA
template strand (thru base pairing):
T in DNA is transcribed as A in RNA
G in DNA is transcribed as C in RNA
A in DNA is transcribed as U in RNA
C in DNA is transcribed as G in RNA
Structure of RNA
a. Ribosomal RNA, rRNA
- is associated with a number of different proteins
as components of the ribosome (complex as
site for protein synthesis)
Mitochondria: 23S, 16S and 5S (P) and (E)
Eukaryotic cytosol: 28S, 18S, 5.8S and 5S
- 80% of the RNA in the cell
b. Transfer RNA, tRNA
- smallest RNA molecule (4S)
- serves as adaptor molecule that carries its
specific amino acid to the site of protein
synthesis
- 15% of the RNA in the cell
c. Messenger RNA, mRNA
- carries genetic information from the DNA to the
cytosol, where it is used as the template for
protein synthesis
- 5% of the RNA in the cell
tRNA
structure
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 BIO20 Introduction to Biomimetic Engineering and Component Design
TRANSCRIPTION OF PROKARYOTIC GENES
Properties of RNA polymerases
RNA polymerases recognize nucleotide sequence
at the beginning of a length of a DNA to be
transcribed. RNA synthesized from its 5’ end to its
3’end antiparallel to its DNA template.
Core enzymes – responsible for the 5’ – 3’ RNA
polymerase activity
Holoenzymes – enables polymerase to recognize
promoter region on the DNA.
Termination factor – RNA polymerase recognizes
regions that signal termination of transcription.
Steps in RNA synthesis:
Initiation – preparation of the DNA template for
RNA synthesis
RNA polymerase holoenzyme binds to a region of
the DNA upstream from the transcribable unit called
promoter regions. For prokaryotes the following are
promoter regions:
Pribnow box which is a stretch of 6 nucleotides
found approximately -7 to -10 upstream with a
consensus sequence of TATAAT
The –35 sequence located about 35 bases to the
left of transcription start site (-35 nucleotide) with a
consensus sequence TTGACA
Elongation – RNA polymerase core enzyme
synthesize transcript of the DNA sequence and
releases the sigma subunit. It synthesizes RNA
from 5’ – 3’ direction anti-parallel to template DNA
strand.
Binding of RNA polymerase to DNA template result
to local unwinding of the helix. Positive supercoils
are relaxed by gyrase, negative supercoils are
relaxed by topoisomerase I.
Termination
-independent termination – requires RNA
transcript to form a stable hairpin turn where at the
stems contain G-C rich bases.
RNA transcripts contain string of U’s. Bonding of U
with A is weak which facilitates separation of newly
synthesized RNA from its DNA template.
-dependent termination – requires an RNA-
dependent ATPase activity. A -factor binds at the
termination region of the DNA that signals RNA
polymerase a cease on transcription.
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Central Dogma of Molecular Genetics
TRANSCRIPTION OF EUKARYOTIC GENES
Promoter regions:
Hogness box – stretch of 4 nucleotides located
approximately at -25 base with a consensus
sequence TATA
CAAT box – stretch of 4 nucleotides located
approximately at -50 to -110 base with a consensus
sequence of CAAT
RNA Polymerases of eukaryotic cells
RNA pol I – synthesize precursor of large rRNA’s in
the nucleolus.
RNA pol II – synthesize precursor of mRNA’s that
are translated to produce proteins.
Promoters for class II genes - recognition sites:
consensus sequence 25 nucleotides upstream of
the transcription start site, TATA or Hogness box;
second consensus sequence, CAAT box.
Enhancers – increase rate of initiation of
transcription of RNA pol II. The bind proteins that
interact with transcription factors bound to a
promoter.
Inhibition of RNA pol II by -amanitin forms tight
complex with the polymerase inhibiting mRNA
synthesis.
3. RNA polymerase III – produces small RNAs
including tRNAs, 5S rRNA, and some snRNAs.
Transcription factors
Initiation complex – minimal complex capable of
initiating transcription: the TATA binding protein
(TBP) and TFIID, a multi-subunit structure
incorporating both TBP and TATA binding
associated factors (TAFs).
Control elements - DNA sequence that binds a
transcription factor. Because the transcription of
genes for proteins must be both tissue specific and
developmentally specific, it requires a great deal of
regulation. These are proteins synthesized on other
genes that enhance or repress the transcription of
the gene in question.
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 BIO20 Introduction to Biomimetic Engineering and Component Design
Central Dogma of Molecular Genetics

Post-transcriptional Modification (Eukaryotes) c. RNA Splicing - After being capped, the pre-
A. Ribosomal RNA mRNA becomes complexed with a number of
- intermediate-sized pieces of rRNAs are small nuclear ribonucleoprotein particles
trimmed to produce the required rRNA (snRNPs), which are themselves complexes of
species. small nuclear RNAs (snRNAs) and special
splicing enzymes. The snRNP--pre--mRNA
complex is called a spliceosome. snRNAs
recognize and bind intron--exon splice sites by
Ribosomal RNA gene means of complementary sequences. Excision
of a single intron involves assembling and
RNA pol I disassembling a spliceosome. The sequence of
reactions can be summarized as follows:
45S
1. It begins with the attachment of the U1
(28S) (5.8S) (18S) snRNP to the G site at the 5' end of the intron.
2. The U2 snRNP then attaches at the branch
cleavage by RNases site.
3. Assembly of the spliceosome continues,
including the addition of several more snRNPs,
(28S) (5.8S) (18S) 4. The lariat loop in the intron is formed and the
Ribosomal RNAs two exons are joined. Splicing has now been
accomplished, and the products--a ligated
B. Transfer RNA mRNA and a looped intron--are released. As
-modifications include addition of –CCA the spliceosome disintegrates, the looped intron
sequence by nucleotidyltransferase on the 3’- is degraded, and the mRNA is exported from
terminal end of tRNAs the nucleus.
C. Messenger RNA
a. 5’ Capping - the first modification occurs at the
5' end of the pre-mRNA. A GTP residue is
added in reverse orientation and forms,
together with the first two nucleotides of the
chain, a structure known as a cap. The cap is
"decorated" by the addition of methyl groups to
the N-7 position of the guanine and to one or
two sugar hydroxyl groups of the cap
nucleotides. The cap structure serves to
position the mRNA on the ribosome for
translation.
b. Addition of poly-A tail – adenine nucleotide is
added after transcription by poly-A polymerase.
This modification helps stabilize the mRNA and
facilitate their exit from the nucleus.

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 BIO20 Introduction to Biomimetic Engineering and Component Design
Central Dogma of Molecular Genetics

TRANSLATION Process of translation:

- conversion of the information in the nucleic acid
sequence to polypeptides of a specific amino acid
sequence.
- requires a system to bring the mRNA together
with the translating molecules (tRNAs).
- message in mRNAs is the sequence of nucleotide
bases and is always read in the 5' to 3' direction.
- each tRNA molecule for a particular amino acid
contains a three nucleotide sequence, called an
anticodon, which is complementary to the codon
for that amino acid.
- specific enzyme aminoacyl tRNA synthetase
catalyzes the attachment between each tRNA
and its corresponding amino acid.

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 BIO20 Introduction to Biomimetic Engineering and Component Design
Central Dogma of Molecular Genetics

Termination of Translation
THE STANDARD GENETIC CODE
Marshall Nirenberg and Philip Leder in 1964

In the genetic code, nucleotide triplets specify an

amino acid.
43 = 64 possible word codes

61 out of 64 possible word codes for

specific amino acids– referred to as codons

3 codons are stop signals

Characteristics of the genetic code:

1. Specificity – specific codon always code for the

same amino acid

2. Redundancy – an amino acid may have more

than one codon coding for it

3. Non-overlapping and commaless – code is read

from a fixed starting point as a continuous

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 BIO20 Introduction to Biomimetic Engineering and Component Design
Central Dogma of Molecular Genetics

DNA MUTATION AND DNA REPAIR

B. Deletion Mutation – removal of one or more
Mutation is the alteration in gene sequence. nucleotides in the DNA.
 Hereditary (mutation on the germline)
]

Consequence of Deletion:
 Change in structure of nucleotide sequence of the FRAMESHIFT MUTATION – a shift in the
DNA reading frame, which produces a non-functional
protein
a. Mutation can be spontaneous or induced.
ATT GGC AAA GCA TCA AAC TTA GGG CCC
b. Mutation according to size:
Point mutation deletion of G at
Gross Chromosomal mutation codon 4
Effects: ATT GGC AAA CAT CAA ACT TAG GGC CC…
 Lethal --- reading frame was changed
 Branching in the evolutionary tree or specie
diversity C. Insertion – addition of one or more nucleotides
in the DNA
Point Mutation
Consequence of Deletion:
A. Substitution mutation FRAMESHIFT MUTATION – a shift in the
- single-base mutations, substitution of one or reading frame, which produces a non-functional
more nucleotides by the same number of protein
different nucleotides.
 Deletion and Insertion Mutations are
Types: Transition and Transversion usually caused by Replication Slippage
Transition
purine to purine D. Exon Skipping – results from mutation at the
A  G spliced site

pyrimidine to pyrimidine Splicing of an intron requires an essential

C  T signal, for example, “GT…….AG”. If the spliced
acceptor site AG is mutated, the splicing
Transversion machinery will look for the next acceptor site.
purine to pyrimidine (vice-versa) As a result, the exon between two introns is
A  C G  C also removed.
A  T G  T
Exon 1 Exon 2 Exon
3
Consequences: GT AG GT AG
1. SILENT MUTATION – no effect on the amino Intron 1 Intron 2
acid sequence of the protein.
Ex: CAG  CAA A to C mutation
Gln  Gln

2. MISSENSE MUTATION – results in the Exon 1 Exon 2 Exon

3
incorporation of a different amino acid in the GT CG GT AG
protein. The protein may be functional, partially Intron 1 Intron 2
functional or non-functional.
CAG  CAC Spliced Message:
Gln  His Exon 1 Exon 3

3. NONSENSE MUTATION – results in the

premature appearance of the stop codon
resulting to a shortened protein that is likely to Removed Introns:
be non-functional.
CAG  TAG Exon 2
GT CG GT AG
Gln  Stop Intron 1 Intron 2

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 BIO20 Introduction to Biomimetic Engineering and Component Design
SPONTANEOUS MUTATION
- likely mutations that occur because of the
following:
1. During DNA Replication
2. Insertion or deletion during replication slippage
3. Tautomer mispairing
INDUCED MUTATION
- are mutations that were acquired from the
environment (environmental factors).
Physical Agents
- mutagens in the form of high energy radiation
 UV Radiation – leads to the formation of
dimmers which can block DNA replication
or interfere with base pairing.
 X-rays – leads to single and double-
stranded DNA breakage or may lead to
formation of hydroxyl radicals from water.
Chemical and Mutagens
 Base Analogues
- have similar structures to the DNA bases
which undergo tautomerism enabling to
cause tautomer mispairing
Example: 5-bromouracil
 Alkylating agents
- adds a methyl or ethyl group to a base
- the largest class of potential mutagens
present in man’s environment
Example: N-nitrosoamines (in cigarette smoke)
When N-nitrosoamine is in the liver, it is
metabolized by liver enzymes to form alkylating
agents which can attack guanine. If alkylation
occurs, an apurinic site is produced. During
replication, an apurinic site may be ignored
resulting in a deletion in the daughter strand or a
base selected at random and place in the daughter
strand.
 Deaminating agents
- removes amino groups from a base
Example: Sodium nitrite (used as preservative,
color enhancer and color fixative bacon, smoked
fish, tocino, etc.)
When ingested, NaNO2 is converted to nitrous acid
in acidic conditions. HNO2 removes functional
groups from Adenine, Guanine and Cytosine. Like
deamination of adenine, will result in the formation
of hypoxanthine, a base analogue of guanine.
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Central Dogma of Molecular Genetics
 Intercalating agents
- contains a cyclic system that can interact with
the bases of the DNA
- it physically binds to the bases by inserting
itself between adjacent base pairs because of
its planar structure. This affects the opening
of the DNA during replication or transcription.
Examples:
Benzo-a-pyrene (found in cigarette smoke)
Benzene (an organic solvent)
Aflatoxin (a metabolic product of molds in peanuts,
oils and grains)
 Viral agents
- some viruses contain oncogenes which can
be activiated once they have inserted their
DNA in the host’s genome.
- When virus insert their DNA into the host’s
genome, the sequence of the bases of the
host DNA may be altered or certain
destructive genes can be activated.
References:
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell
Biology, 5th edition. New York: W. H. Freeman;
2004.
Matthews, van Holde and Ahern, Biochemistry 3rd edition
Lehninger, Nelson and Cox (1993). Principles of
Biochemistry, 2nd edition, Worth Publishers, New
York
Starr and Taggart, Biology. The Unity and Diversity
of Life, 10th edition, Wadsworth Group, Thomson
Learning, Inc., California, 2010.
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