Course MDBC 204
Instructor: Dr. A.R. Al-Mutairy
SOFTWARE OF LIFE
Genes: Replication and Expression
Textbook: Biochemistry by Stryers(4th ed.)
Chapters 4,5,6,31,32, 33, 34 & 35
B. DNA Replication and Repair
C. Gene Rearrangements
D. RNA Synthesis (Transcription)
E. Protein synthesis (Translation)
F. Protein Targetting
• Until 1944 it was assumed that chromosomal proteins
carry the genetic information.
• In 1944 Avery et al. Concluded that DNA carry the genetic
information based on Griffith (1928) experiments on rough
and smooth pneumococci.
• DNA = Deoxyribonucleic acid, RNA = Ribonucleic acid
• DNA & RNA are polynucleotides
• A mononucleotide consists of: A nitrogen base + Sugar +
• A deoxyribonucleotide = A nitrogen base + Deoxyribose +
• A Ribonucleotide = nitrogen base + ribose + PO4
• A nucleoside = nitrogen base + sugar
• DNA & RNA are the molecules of Heredity
5 major bases (A, G, C, T & U, Alphabet of life)
• The discovery that genes are made up of DNA (Avery.
et. al. 1944) and its D-helical structure (Waston-Crick
1953). Triggered a revolution in Biology (including
• Based on X-ray Crystallographic studies, Watson and
Crick in 1953 proposed the following model for 3-
Dimensional Structure of DNA:
1. DNA consists of 2 helical polynucleotides which run
in opposite directions.
2. The bases are inside, PO4 and d-ribose outside d-helix.
3. Diameter of he helix is 20A˚. Bases separated by
3.4A˚ and related by a rotation of 36 degree (10
residue per turn).
4. The 2 chains are held by H-bonding between
complementary bases (A - T & G - C).
G - C - Base-pair) Cross sectionA - T base-pair
Semi-conservative replication of DNA:
Each strand act as a template on which another
complementary strand is synthesised.
Reversible melting (Denaturation):
H - bonds between A - T & G - C can be broken by heat,
alkalis and acids lead to separation (unwinding) of 2-chains.
Tm: Temp. at which half of the helical structure is broken.
Physical Properties of DNA:
Size: Length of DNA in E.coli 1.4 mm
(4 million base-pairs) in Human 1
Relaxation of supercoiled DNA
by Topoisomerase I
5 min 30 min
Mechanism for DNA replication:
• In 1958, Kornberg isolated DNA polymerase from E.coli: The
Enzyme can invitro replicate DNA and requires:
1. 4 precursors: dATP, dTTP, dGTP and dCTP
2. A primer with free 3`-OH; (3) DNA template
DNA Polymerase I: The enzyme has 3 functions
5` 3` Exonucleases activity
3` 5` Exonucleases activity
DNA Polymerase I: (continue)
1) 3` 5` Exonuclease activity (proof reading):
- Function to remove mismatched nucleotide at 3`- end.
2) 5` 3` Exonuclease:
For removal of primer and it participate in removal of
pyrimidine dimers formed by exposure of DNA to UV
3) Polymerase activity:
DNA Polymerase II and III: similar to I in:
a) they catalyse a tempelate - directed synthesis of DNA.
b) A primer with free 3`-OH is needed fo DNA synthesis.
c) The direction of DNA synthesis is 5` 3` direction.
d) They have 3` 5` exonuclease activity.
Highlights of DNA replication: in E.coli
1- Unwinding of DNA, is coupled to replication. Circular DNA
maintain its circular structure.
2- Origin of replication: at a fixed site (oriC, in E.coli)
3- Synthesis of leading and lagging strands
Steps of DNA Replication (in E.coli)
a) dnaA protein detects and bind to oriC.
b) A complex of 3 proteins: dnaA, B and C bend and
open the d-helix (dna B is a helicase).
c) Single strand parts are stabilized by S.S.B. Protien.
d) DNA-Gyrase forms -ve supercoil to facilitate
unwinding and replication.
I- Unwinding the Origin Site (oriC):
II- Synthesis of RNA-primer by primase ( 5 nucleotide
III- DNA-Polymerase III start synthesis of DNA
The leading strand is synth. Continuously, while the
lagging strand is synth., as Okazaki fragments, both in the
5` 3` direction
IV. Gaps between Okazaki fragments are filled by DNA
polymerase I - then joined by DNA ligase.
Processive sliding Clamp (β) DNA in clamp
1. Insertion 2. Deletion
3. Substitution (point mutation) most common:
a) Transition: Substitution of one purine by another
purine or pyrimidine by the other.
b) Transversion: Purine Pyrimidine
Repair of mutation: Examples
1. The complementary structure of DNA ensures the damage in one strand will
be repaired in later replication.
2. Removal of pyrimidine dimers (caused by UV)
3. Mismatch Repair: (in E.coli)
Many cancers are caused by defective Repair of
1. Xeroderma Pigmentosum:
• Skin very sensitive to light lead to skin cancer. Cause is
defective uvr ABC enzyme.
2. Heriditary nonpolyposis Colorectal cancer (HPCC)
• Result from defective mimtach repair (1 in 200 people).
Mutation in 2 genes hMSH2 and hMLH1 account for most
cases of HPCC.
• hMSH2 and hMLH1 in human are counterparts of MutS &
MutL in E..coli.
D) RNA Synth. (Transcription) & Splicing
Flow of genetic information:
DNA (genes) mRNA Proteins
Structure of RNA:
A polymer of ribonucleotides joined by 3` 5`
Phosphodiester bonds. Similar to DNA except:
i) It is single strand ii) Contains Uracil (no thymine)
iii) Contains ribose instead of deoxyribose iv) No Nuclease activity
chance of mistake 10-4
. Compare with replication of DNA 10-10
Types of RNA in E.Coli
Type % amount # of Nucleotides
Ribosomal RNA (rRNA) 80 120-3700
Transfer RNA (tRNA) 15 75
Messenger RNA (mRNA) 5 1200-5000
Function of RNA:
a) mRNA: A template for protein synth. (Translation)
b) tRNA (carry amino acids in an activated form to the ribosome for
peptide bond formation.
c) rRNA: 5S, 16S&23S rRNAs play a Central role in protein synthesis.
Ribosome: Site of protein synthesis
• A complex assembly of rRNA and ~50 diff. Proteins.
In E.coli, all RNAs are synth. By RNA-polymerase which requires:
i) A template (DNA), ii) ATP, GTP, UTP and CTP.
• RNA-polymerase catalyse both initiation and elongation of RNA.
RNA-Polymerase takes instructions from a DNA template
Transcription begins near promotor sites and ends at terminator sites.
tRNA is the adoptor molecule in protein synthesis.
The Genetic Code
• Amino acids are coded by groups of 3 bases.
i) The code is degenerate, i.e. most a.as are coded by more
than one codon (triplet).
ii) The code is non-overlapping.
iii) The sequence of bases is read from a fixed point.
Mechanism of RNA Synth. (in E.coli)
• δ- subunit of RNA-polymerase recognize and binds to promoter site of DNA
• A strong promoter site: one very close to the consensus sequence. (Fast
transcription one every 2 sec.
• Weak promoter: seq. Deviate from concensus Seq. (slow transcription ~ 10
• RNA - polymerase unwinds 2-turns of template DNA.
• RNA-chain start with G or A, in 5` 3` direction
2. Elongation: start after first bond
• δ-subunit dissociate.
• -Newly synth. RNA form d. helix with template strand.
• An RNA hair pin followed by several U residue
lead to termination of transcription.
• The hair pin structure is specified by a
palindromic G-C-rich followed by A-T-rich
region on template.
• The RNA-DNA hybrid dissociate and Nascent
Rifampicin & actinomycin antibiotics inhibit
Transcription in Eukaryotes
• Transcription occurs inside organells
• Transcription and translation are separated in time and space which
enables eukaryotes to regulate gene expression more intricately
which contribute to the richness of eukaryotic form and function.
• Primary RNA mRNA.
i) A cap is added at end and a poly A tail.
ii) Eukaryotic genes contain coding sequences (Exons) & non-coding
(intervening) sequences (introns). Introns of primary transcripts are
cut off and exons are spliced to form mRNA.
RNA in Eukaryotes is synth. By 3 kinds of RNA-polymerase (I, II & III)
Many transcription factors (proteins) interact with promoter - sites.
Enhancer sequences can stimulate Transcription at start sites thousands
of bases away.
Splice sites are specified by sequences at ends of introns.
• Normal cells do not contain tRNAs with anticodons complementary to UAA, UGA or UAG (stop codons).
• Instead of these codons are recognized by release factors RF1 recognizes UAA & UAG. RF2 recongizes UAA
• Binding of a release factor to a stop codon in the A site activates the hydrolysis of the bond between the
polypeptide and the tRNA in the P-site. The detached polypeptide leaves the ribosome, followed by tRNA and
Antibiotics: Puromycin, Streptomycin, Tetracyclin, Chloramphenicol & Erythromycin inherit protein
3. Elongation Stage: