This document provides an overview of the course MDBC 204 which covers topics related to genes, DNA replication and expression, RNA synthesis and protein synthesis. It discusses the key discoveries in genetics including the identification of DNA as the genetic material and the discovery of its double helix structure. The summary also outlines the basic mechanisms of DNA replication, transcription, translation and protein targeting in both prokaryotes and eukaryotes.

1. 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
A. Overview
B. DNA Replication and Repair
C. Gene Rearrangements
D. RNA Synthesis (Transcription)
E. Protein synthesis (Translation)
F. Protein Targetting

2. 2

3. 3
A-Overview:
• 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 +
PO4
• A deoxyribonucleotide = A nitrogen base + Deoxyribose +
PO4
• A Ribonucleotide = nitrogen base + ribose + PO4
• A nucleoside = nitrogen base + sugar
• DNA & RNA are the molecules of Heredity

4. 4
5 major bases (A, G, C, T & U, Alphabet of life)

5. 5

6. 6
• 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
medicine)
• 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).

7. 7
G - C - Base-pair) Cross sectionA - T base-pair

8. 8
Semi-conservative replication of DNA:
Each strand act as a template on which another
complementary strand is synthesised.

9. 9
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
meter

10. 10
Relaxed DNA
Supercoiled
Relaxation of supercoiled DNA
by Topoisomerase I
5 min 30 min
0

11. 11
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

12. 12
DNA Polymerase I: The enzyme has 3 functions
5` 3` Exonucleases activity
3` 5` Exonucleases activity

13. 13
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
light.
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.

14. 14
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

15. 15
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.

16. 16
Replication Fork:
Processive sliding Clamp (β) DNA in clamp

17. 17
Genetic Mutation
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

18. 18
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)

19. 19
Many cancers are caused by defective Repair of
Mutation:
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.

20. 20
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
 →
ionTranscript
 →
nTranslatio

21. 21
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.

22. 22
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.
Hair Pin
Termination
Signal
tRNA

23. 23
The Genetic Code
• Amino acids are coded by groups of 3 bases.
• Features:
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.

24. 24

25. 25
Mechanism of RNA Synth. (in E.coli)
1. Initiation:
• δ- subunit of RNA-polymerase recognize and binds to promoter site of DNA
template.
• 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
min).
• 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.

26. 26
3. Termination:
• 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
RNA released.
Rifampicin & actinomycin antibiotics inhibit
transcription.

27. 27
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.
 →
extensive
processing
Exon
Exon

28. 28
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.

29. 29

30. 30

31. 31
E) Protein Synthesis
(Translation)
1. Amino acid Activation
nTerminatio
ElongationinitiationChainactivationa.a.
↓
→→
2. Initiation
Synthetase RNAAminoacylttRNAa.a.
tRNAa.a.
 →+

32. 32
Initial Stage
Steps of Chain Elongation

33. 33
Initiation Stage

34. 34
4. Termination:
• 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
& UGA.
• 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
mRNA.
Antibiotics: Puromycin, Streptomycin, Tetracyclin, Chloramphenicol & Erythromycin inherit protein
synthesis
3. Elongation Stage:

35. 35