The nucleotide structure ,consists of
the nitrogenous base ,attached to the 1’ carbon of deoxyribose
,
the phosphate group attached to the 5’ carbon of deoxyribose
,
a free hydroxyl group (-OH) ,at the 3’ carbon of deoxyribose,1. DNA HELICASES,
to separate the strand,
2. GYRASE (Topoisomerases),
unwind the supercoil,
3. Single strand binding protein (SSBP)
, activity of helicase,
keep two strand separate,
protect DNA from nuclease degradation,
release after replication,
2. DNA Structure
DNA is a nucleic acid
The building blocks of DNA are nucleotides, each composed of:
A 5-carbon sugar called deoxyribose
A phosphate group (PO4)
A nitrogenous base
Adenine, Thymine, Cytosine, Guanine
3. The nucleotide structure consists of
the nitrogenous base attached to the 1’
carbon of deoxyribose
the phosphate group attached to the 5’
carbon of deoxyribose
a free hydroxyl group (-OH) at the 3’
carbon of deoxyribose
4.
5. Nucleotides are connected to each other to form a long chain
phosphodiester bond: bond between adjacent nucleotides
formed between the phosphate group of one nucleotide and the 3’ –OH of
the next nucleotide
The chain of nucleotides has a 5’ to 3’ Orientation
7. Determining the 3-dimmensional structure of DNA
involved the work of a few scientists
Erwin Chargaff determined that
amount of adenine = amount of thymine
amount of cytosine = amount of guanine
This is known as Chargaff’s Rules
8. James Watson and Francis Crick, 1953
deduced the structure of DNA using
evidence from Chargaff, Franklin, and
others
proposed a double helix structure
9. The double helix consists of:
2 sugar-phosphate backbones
nitrogenous bases toward the interior of the
molecule
bases form hydrogen bonds with
complementary bases on the opposite
sugar-phosphate backbone
10. The two strands of nucleotides are antiparallel to each other
one is oriented 5’ to 3’, the other 3’ to
5’
The two strands wrap around each other to create the helical shape of
the molecule
11.
12. In these grooves,
proteins can interact
specifically with
exposed atoms of the
nucleotides (usually by
H bonding) and
thereby recognize and
bind to specific
nucleotide sequences
without disrupting the
base pairing of the
double-helical DNA
molecule
13.
14. A B Z
Shape Broadest Intermedi
ate
Narrowest
Rise per
base pair
2.3 Å 3.4 Å 3.8 Å
Screw
sense
Right
handed
Right
handed
Left handed
Base per
turn
11 10.4 12
Glycosidic
Bond
Anti Anti Alternating Anti
and syn
Comparison of A , B and Z forms of DNA
15. STEPS INVOLVED IN DNA REPLICATION
1. Identification of the origins of replication
2. Unwinding (denaturation) of dsDNA to provide an
ssDNA template
3. Formation of the replication fork; synthesis of RNA
primer
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of the
newly synthesized DNA segments
6. Reconstitution of chromatin structutre
15
16. DNA replication is the process
where an entire double-stranded
DNA is copied to produce a
second, identical DNA double
helix
Meselson and Stahl concluded
that the mechanism of DNA
replication is the semiconservative
model
Each strand of DNA acts as a
template for the synthesis of a
new strand
16
Q. During which phase
of the cell cycle
does DNA replicate?
17. FACTORS NEEDED FOR REPLICATION
1. DNA HELICASES
to separate the strand
2. GYRASE (Topoisomerases)
unwind the supercoil
3. Single strand binding protein (SSBP)
activity of helicase
keep two strand separate
protect DNA from nuclease degradation
release after replication
17
18. 4. PRIMOSOME
Complex Protein (contains)
primase
helicase (dna B)
act as primer for DNA synthesis
removed by polymerase I
18
19. 5. DNA SYNTHESIS CATALYZED BY DNA POLYMERASE
III
New strand synthesis is catalyzed by DNA poly III
5' 3' direction
Antiparallel to parent template
dNTPs are added one after another to 3‘
Newly synthesized strand nucleotide is
complementary to parent DNA strand
19
20. 6. DNA LIGASE:
It catalyses the formation of phosphodiester linkage
between small fragment of DNA
7. TERMINATION
Termination utilization sub (Tus) protein binds to
termination sequence prevent helicase (dna B)
protein
Unwinding of DNA helix is stopped
20
21. Initiation of Replication
Initiation starts at a site called origin of replication
The origin of replication in E. coli is termed oriC
origin of Chromosomal replication
Important DNA sequences in oriC
AT-rich region
DnaA protein binds with the site of origin causing the double
stranded DNA to separate
The dsDNA is unwound by Helicase (DnaB)
This generates positive supercoiling ahead of each replication
fork
21
22. Initiation of Replication
DNA gyrase: relieves tension from the unwinding of
the DNA strands during bacterial replication
It cuts nicks in both strands of DNA, allowing them to
swivel around one another and then resealing the cut
strands
The single stranded binding proteins (SSBs) bind to
the exposed bases to prevent them from annealing
Then short (5 to 50 nucleotides) RNA primers are
synthesized by primase
22
23. These short RNA strands start, or prime DNA synthesis
DNA polymerases can synthesize DNA only in the 5’ to 3’
direction
DNA synthesis is semidiscontinuous and bidirectional
On the leading strand the DNA synthesis is
continuous
On the lagging strand the DNA synthesis is discontinuous
DNA polymerase III adds complimentary nucleotides
(deoxyribonucleoside triphosphates) in the 5’ to 3’ direction, using
RNA points
The segments arprimers as starting e called Okazaki fragments
23
24. Building Complimentary
Strands In prokaryotes, there are 3
enzymes known to function in
replication & repair
DNA polymerase I, II & III
In eukaryotes, there are 5
enzymes known to function
in replication & repair
DNA pol α, β, γ, δ,ε
24
26. Building Complimentary Strands in
prokaryotes
RNA primers are synthesized by primase and are temporary
The leading strand (uses 3’-5’ template) is synthesized
continuously
The lagging strand (uses 5’-3’ template) is synthesized
discontinuously in short fragments
26
27. Building Complimentary Strands in
prokaryotes
DNA polymerase I removes the RNA primers from
the leading strand and fragments from the lagging
strand and replaces them with the appropriate
deoxyribonucleotides
27
29. Building Complimentary Strands in
prokaryotes
DNA ligase joins the Okazaki fragments into one
strand on the lagging strand of DNA through the
formation of a phosphodiester bond
29
31. Building Complimentary
Strands in prokaryotes
As the 2 new strands of DNA are
synthesized, 2 double stranded DNA
molecules are produced that automatically
twist into a helix
31
32. PROOF READING
Fidelity of replication is important is done by DNA Polymerase II
DNA Polymerase III – beside synthesis also perform proof reading activity
(3’ 5’ exonuclease activity)
Check during synthesis
Correction
32
33. PROCESSIVITY
The average number of nucleotides added before a polymerase
dissociates defines its processivity
DNA polymerases vary greatly in processivity; some add just a few
nucleotides before dissociating, others add many thousands
33
34. EUKARYOTIC REPLICATION
Multiple origin site
Multiple replication bubbles
Enzymes – 5 types
DNA polymerase - , , , and
DNA Poly = Synthesis of RNA primer (both strand)
Responsible for initiation
Poly = Repair of DNA
Poly = Replication of mitochondrial DNA
Poly = Replication of leading & lagging strand
Proof reading
Poly = Lagging strand
34
35. Transcription
RNA synthesis, or transcription, is the process of transcribing DNA
nucleotide sequence information into RNA sequence information.
RNA synthesis is catalyzed by a large enzyme called RNA polymerase.
It takes place in three stages: initiation, elongation, and termination.
36. RNA polymerase performs multiple
functions in transcription
1.It searches DNA for initiation sites, also called promoter sites.
2.It unwinds a short stretch of double-helical DNA to produce a single-
stranded DNA template from which it takes instructions.
3.It selects the correct ribonucleoside triphosphate and catalyzes the
formation of a phosphodiester bond.
4.It detects termination signals that specify where a transcript ends.
5.It interacts with activator and repressor proteins that modulate the rate
of transcription initiation over a wide dynamic range.
37. Transcription cycle
(1) Template binding: RNA polymerase (RNAP) binds to DNA
and locates a promoter (P) melts the two DNA strands to
form a preinitiation complex (PIC).
(2) Chain initiation: RNAP holoenzyme (core + one of multiple
sigma factors) catalyzes the coupling of the first base
(usually ATP or GTP) to a second ribonucleoside
triphosphate to form a dinucleotide.
(3) Chain elongation: The fundamental reaction of RNA
synthesis is the formation of a phosphodiester bond. The 3 -
hydroxyl group of the last nucleotide in the chain
nucleophilically attacks the phosphate group of the incoming
nucleoside triphosphate with the concomitant release of a
pyrophosphate
(4) Chain termination and release: The completed RNA chain
and RNAP are released from the template. The RNAP
holoenzyme re-forms, finds a promoter, and the cycle is
repeated.
39. The strand that is transcribed or copied into an RNA
molecule is referred to as the template strand of the
DNA.
The other DNA strand is frequently referred to as the
coding strand of that gene.
40. Transcription starts at promoters on the DNA template.
Promoters are sequences of DNA that direct the RNA
polymerase to the proper initiation site for
transcription.
Two common motifs are present on the 5 (upstream)
side of the start site.
They are known as the -10 sequence and the -35
sequence because they are centered at about 10 and
35 nucleotides upstream of the start site.
47. Translation
The four letter alphabet of nucleic acid is
translated into entirely different 20 alphabets of
proteins
48. Translation Requirements
mRNA- Template
tRNA- Adaptor molecule
Ribosome- Molecular machinery
Amino acids- Precursors of protein
Enzyme and translation factors
Energy- ATP and GTP
49. mRNA
Template
Nucleotides are arranged in triplet
of bases – Codons
Protein coding region of each
mRNA is composed of a
contiguous, non-overlapping
string of codons called an
opening reading frame (ORF)
Begins with start codon and end with stop codon
51. Activation of tRNA
Some tRNA recognize more than one codon
Codon-anticodon interaction – Complementary base pairing and
antiparallel
First two bases of codon and anticodon pair in standard
way
Codon differ in 1st two bases are read by different tRNA
Eg. UUA and CUA – Leucine
1st base of anticodon determine number of codon read by
one tRNA
C or A – One
U or G – Two Eg. UUA & UUG and UUU & UUC
I – three Eg. GCU, GCC, GCA (Ala)
52. rRNA
Molecular machinery- Protein
Synthesis
Ribonucleoprotein assemblies
2/3rd of mass rRNA
Recognition of start signal –
16SrRNA
Peptidyl transferase – 23SrRNA
56. Activation
Activation of amino acids
Amino-acyl tRNA synthetases, ATP and tRNA
Form ester with either 3’-OH or 2’-OH of terminal
Adenine residue of tRNA
Two equivalent ~P used
Ester linkage and drive the reaction
Aminoacyl-tRNA synthatases belong to 2
Classess
Class I acylate at 2’-OH and for Larger and more
hydropobic amino acid
Class II acylate at 3’-OH (Except Phe-tRNA)
58. In prokaryotes, Shine-
Dalgarno sequences
recognized by an initiation
complex
consisting of a Met amino-
acyl tRNA,
Initiation Factors (IFs) and
the small ribosomal subunit
59. GTP hydrolysis by IF2 coincident
with release of the IFs and binding
of the large ribosomal subunit
leads to formation of a complete
ribosome, on the mRNA and ready
to translate.
60. Nucleotide sequence in mRNA signal where to start
protein synthesis
The translation of an mRNA begins with codon AUG.
In eukaryotes cap and in prokaryotes Shine - Dalgarno
sequence tell ribosome where to begin searching for
the start of translation
61. A U G G G C U U AAA G C A G U G C A C G U U
A ribosome on the rough endoplasmic
reticulum attaches to the mRNA molecule.
ribosome
62. A U G G G C U U AAA G C A G U G C A C G U U
It brings an amino acid to the first three
bases (codon) on the mRNA.
Amino acid
tRNA molecule
anticodon
U A C
A transfer RNA molecule arrives.
The three unpaired bases (anticodon)
on the tRNA link up with the codon.
63. A U G G G C U U AAA G C A G U G C A C G U U
Another tRNA molecule comes into
place, bringing a second amino acid.
U A C
Its anticodon links up with the second
codon on the mRNA.
64. A U G G G C U U AAA G C A G U G C A C G U U
A peptide bond forms between the
two amino acids.
Peptide bond
65. A U G G G C U U AAA G C A G U G C A C G U U
The first tRNA molecule releases its amino
acid and moves off into the cytoplasm.
66. A U G G G C U U AAA G C A G U G C A C G U U
The ribosome moves along the mRNA to
the next codon.
67. A U G G G C U U AAA G C A G U G C A C G U U
Another tRNA molecule brings
the next amino acid into place.
68. A U G G G C U U AAA G C A G U G C A C G U U
A peptide bond joins the second
and third amino acids to form a
polypeptide chain.
69. A U G G G C U U AAA G C A G U G C A C G U U
The polypeptide chain gets longer.
The process continues.
This continues until a termination
(stop) codon is reached.
The polypeptide is then complete.
71. mRNA
Contains initiation and termination signal
Collection of codons – Genetic code
Degeneracy
Unambiguous
Non overlapping
No punctuation
Nearly Universal
Synonyms
Codons decode the same amino acids
UUU and UUC – codon for Phe
AUG – Met and UGG- Trp
Two (rest), Three (Ile), Four (TGVPA) and Six
Codons (SLR)
72. XYU and XYC always code same amino acid
XYA and XYG usually code same amino acid
