DNA & RNA

The central dogma of life (or molecular biology)
presented in the form of conventional and current
conceps
The term ‘dogma’ isa
misnomer, introduced
by Francis Crick at a
time when little
evidence supported
these ideas, now the
dogma has become a
well-established
principle.

Structure of DNA
DNA is a polymer, based on D-deoxyribose –
deoxyribonucleotides (deoxynucleotides) – and
phosphoric acid.
It is composed of monomeric units –
deoxyadenylate (dAMP),
deoxyguanylate (dGMP),
deoxycytidylate (dCMP),
deoxythymidylate (dTMP).

Chargraff’s rule
(or rule of molar equivalence between the purines and
pyrimodines in DNA)
Erwin Chargaff observed that DNA had equal numbers
of adenin and thymine residues (A=T), and equal
number of guanin and cytosine residues (G=C) –now
this is known as Chargraff’s rule (or molar
equivalence) between the purines and pyrimodines in
DNA.
Also it was shown that amount of A+C is equal to
G+T

Sponsored
Medical Lecture Notes – All Subjects
USMLE Exam (America) – Practice

DNA double helix (1)
James Watson and Francis Crick
(in 1953 Nobel Prize in 1962)
-DNA is a right handed double helix of
polydeoxyribonucleotide chains (strands) twisted around
each other on a common axis.
-the two strands are antiparallel (i.e. one strand runs in the 5'
to 3' direction while the other in 3' to 5' direction.
-each strand of DNA has a hydrophilic deoxyribose
phosphate backbone (3'-5'-phosphodiester bonds) on the
outside (periphery) of the molecule while the hydrophobic
bases are stacked inside (core)

-the width (or diameter) of double helix is 20 Ǻ (2 nm)
-each turn (pitch) of the helix is 34 Ǻ (3.4 nm) with 10 pairs
of nucleotides, each pair placed at a distance of about 3.4 Ǻ
(0.34 nm)
-the genetic information resides on one of the two strands
known as template strand or sense strand; the opposite
strand is an antisense strand.
-the double helix has (wide) major grooves and (narrow)
minor grooves along the phosphodiester backbone
-proteins interact with DNA at these grooves, without
disrupting the base pairs and double helix

-the two strands are held together by hydrogen bonds
formed by complementary base pairs [A-T – 2 hydrogen
bonds; G-C – 3 hydrogen bonds]; the G≡C is stronger by
about 50% thanA=T
-the hydrogen bonds are formed between a purine and a
pyrimidine only
-the complementary base pairing in DNA gelix proves
Chargaff’s rule (the content of adenine equals to that of
thymine (A=T) and guanine equals to that of cytosine (G=C)

Pairs A=T (2 hydrogen bonds) G=C (3 hydrogen bonds)

The standard A=T and G≡C base pairs have very
similar geometries, and an active site sized to fit one
(blue shading) will generally accommodate the other
Contribution of base-pair geometry to the fidelity of
DNA replication
The geometry
of incorrectly
paired bases
can exclude
them from the
active site, as
occur on DNA
polymerase

Compaction of DNA in eukaryotic
chromosome

Effect of DNA underwinding
(a) A segment of DNA within
a closed-circular molecule
in its relaxed form with
eight helical turns
(b) Remuval of one turn
induces structural strain
(c) The strain is
generally
accommodated by
formation of supercoil
(d) DNA underwinding also
makes the separation of

Supercoils of DNA helix
supercoilind of DNA
a phone cord is coiled like DNA
helix

Positioning of a nucleosome to make optimal use of
A=T base pairs where the histone core is in contact
with the minor groove of DNA helix

Higher order structures provide for the compaction of
chromatin

Nucleosomes
regularly spaced nucleosomes
consists of histone complex
bound to DNA

The 30nm fiber, a higher-order organization of
nucleosomes

Chromatine assembly
(a) relaxed, closed-circular
DNA
(b) (b) binding of a histone core
to form a nucleosome
induced one negative
supercolil
(c) (c) relaxetion of this positive
supercoil by topoisomerase
leaves one net negative
supercoil

Loops of chromosomal DNA attached to a nuclear scaffold.
The DNA in the loops is packaged as 30 nm fibers. Loops are the next [after fiber]level
of DNA organization. Loops often contain groups of genes, with related functions.

A human karyotype
(of a man with a normal 46 XY constitution), in which the chromosomes have been
stained by Giemsa method and aligned according to the Paris Convention)

Linking number (a – Lk=1; b – Lk=6)

Linking number applied to closed-circular DNA
molecules (a – relaxed Lk=200; b – relaxed with a nick [break] in one
strand Lk undefined; c – underwound by two turns, Lk=198)

Defining DNA strands at the replication fork
a new DNA strand (red) is always synthesized in the 5' to 3' direction

Hypothetical scheme for the action of a single-strand binding protein at a
replication fork. The protein is recycled after binding single-stranded
regions of the template and facilitating replication.

DNA replication process
(SSB – single-stranded binding proteins)

Elongation of a DNA chain 25-5

Elongation of a DNA chain by DNA polymerase 25-5

Adding of new nucleotide to DNA strand and result
adding

Replication of the E.coli Chromosome proceeds in
stages
Replication is a process in which DNA copies itself to
produce identical daughter molecules of DNA.
Initiation – is the only phase of DNA replication that is known to be
regulated, the mechanism of regulation is not yet well understood.
Elongantion phase includes two distinct but related operations: leading
strand synthesis and lagging strand synthesis
Termination the two replication occur when replication fork meey a
terminus region containig multiple copies of 20 bp sequence called Ter
(for terminus) – a binding site for protein called Tus (terminus utilization
substance). Tus-Ter complex stop the replication halts. The other
replication fork halts when it meets the first (arrested) fork

Replication of DNA in prokarytes (1)
Replication is a process in which DNA copies itself to
produce identical daughter molecules of DNA.
Replication is semiconservative – half of the original DNA is conserved
in the daughter DNA
Initiation of replication – occurs at a site called origin of replication;
these sites mostly consist of a short sequence of A-T basepair.
Replication bubbles – the two complementary strands of DNA separete
at the site of replication to form a bibble; multiple replication bubblesare
formed in eukaryotis DNA molecules, which is essencial for a rapid
replication process.
RNA primer – short fragment of RNA (about 5-50 nucleotides,variable
with species) for the synthesis of new DNA is required

DNA synthesis is semidiscontinues and bidirectional – The replicationof
DNA occure in 5' to 3' direction, simultraneously, on both strands of
DNA; one of the strand, the leading (continuos or forward) strand – the
DNA synthesis is continuous. On the other strand, the lagging
(discontinuous or retrograde) strand – the synthesis of DNA is
discontinuous. Short pieses of DNA (15-200 nucleotides) are produced
on the lagging strand. In the replication bubble, the DNA synthesis
occurs in both the directions (bidirectional) from the point of origin
Replication fork and DNA synthesis – the separation of the twostrands
of parent DNA results in the formation of a replicationfork
DNA helicase – the enzyme which moves along the DNA helixand
separate the sytands.
Single-stranded DNA binding proteins (DNA helix-destabilizing
proteins) – possess no enzyme activity – bind only to single-stranded
DNA (separated by helicase) keep the two strans separate andprovide
the template for new DNAsynthesis.

DNA syntesis catalysed by DNA polymerase III – the synthesis of anew
DNA strand occur only in 5' to 3' direction. The presence of all the four
deoxyribonucleoside triphospfates (dATP, dGTP, dCTP, and dTTP) is an
essential prerequisite for replication to take place.
The synthesis of two new DNA strands simultaneously, take place in the
opposite direction – away from the replication fork which is
discontinuous.
The incomong deoxyribonucleides are added one after another, to 3'-OH
end of the growing DNAchain
Polarity problem – that lagging strand with 5'-end presents some
problem, as there is no DNA polymrerase enzyme that can catalyse the
addition of nucleotides to the 5'-end (i.e. in 3' to 5' direction) of the
growing chain. This problem is solved by synthesizing this strand as a
serirs of small fragments – are called Okazaki pieces. This Okazaki
pieces are later joined to form a continuous strand of DNA by DNA
polymerase I and DNAligase.

Proof-reading function of DNA DNA polymerase III – it checks the
incoming nucleotides and allows only the correctly matched bases (i.e.
complementary bases). DNA polymerase III edits its mistakes (if any)
and removes the wrongly placed nucleotide bases
Replacement of RNA primer by DNA – the synthesis of new DNA
sytand (on lagging strand) continues till it is in close proximity to RNA
primer. DNA polymerase I removes the RNA primer and take itsposition
and in this position catalyses the synthesis (in 5' to 3' direction) of a DNA
fragment which replases RNAprimer.
DNA ligase catalyse the formation of phosphodiester linkage between
the DNA synthesized by DNA polymerase III and a small fragment of
DNA produced by DNA polymerase I. DNA polymerase IIparticipates
in DNA repairprocess.

Supercoils and DNA topoisomerases – as the double helix of DNA
separates from one side and replication proceeds, supercoils are formed
at the othe side. Type I DNA topoisomerase cuts the single DNAstrand
(nuclease activity) to overcome the problem of supercoils and then
reseals the strand (ligase activity). Type II DNA topoisomerase (also
known as DNA gyrase) cuts both strands and release them toovercome
the problem of supercoils.
DNA topoisomerase are targeted by drugs in treatment of cancers.

Initiators for initiation of DNA replication

DNA polymerasa III
(general view, end view and side view)

DNA synthesis on the leading and lagging strands (1)

Synthesis of Okazaki fragments
(a)At intervals, primase
synthesis an RNAprimer
for a new Okazaki
fragments (synthesis
formally proceeds in the
opposite direction from
fork movement)
(b)Each primer is extended
by DNA polymerase III
(c)DNA synthesiscontinues
until the fragment extends
as far as thr primer of the
previously added Okazaki
fragments. A new primer is
synthesized near the
replication fork to begin the
process again.

Replication Cells of Chromosome proceeds in stages
in Eukaryotic
DNA polymerase α – is responsible for the synthesis ofRNA
primer for both the leading and lagging strands
DNA polymerase β – is involved in the repair of DNA; its function
is comparable with DNA polymerase I found inprokaryotes
DNA polymerase γ – participates in the replication of
mitochondrial DNA
DNA polymerase δ - is responsible for the synthesis replication on
the leading strand of DNA; it also possesses proof-reading activity
DNA polymerase ε – is involved in DNAsynthesis on the lagging
strand and proof-reading function

DNA replication in eukaryotes 1
The replication on the leading (continuous) strand of DNA
is rather simple, involving DNA polymerase δ and a
sliding clamp called proliferating cell nuclear antigen,
which forms a ring around DNA to which DNA
polymerase δ binds. Formation of this ring also requires
another factor: replication factorC.
Replication on the lagging (discontinuous) strand in
eukaryotes after the parental strands DNA are separatedby
the enzyme helicase, a single-stranded DNA binding
protein called replication protein A binds to the exposed
single-stranded template. This strand has been opened up
by the replication fork (a previously formed Okazaki
fragment with an RNAprimer.
The enzyme primase forms a complex with DNA
polymerase α (pol α-primase complex) which initiatesthe
synthesis of Okazaki fragments. Anzyme activityswitched
from primase to DNA polimerase α – which elongates the
primer by the addition of 20-30 deoxyribonucleotides –
thus a short stretch of DNA attached to RNA isformed.

DNA replication in eukaryotes 2
The next step is the binding of replication factor C to the
elongated primer (short RNA-DNA) which serves as a
clamp loader, and catalyses the assebly of proliferating
cell nuclear antigen molecules. The DNA polymeraseδ
binds to the sliding clamp and elongates the Okizaki
fragment to final length of about 100-200bp. By this
elongation, the replication complex approaches the RNA
primer of the previouse Okazaki fragment.
The RNA primer removal is carried out by a pair of
enzymes namly RNase and flap endonuclease I. This gap
created by RNA removal is filled by continued elongation
of the new Okazaki fragment (carrtied out by polymerase
δ). The small nick that remains is finally sealed by
DNAligase.
Eukariotic DNA is tightly bound to histones (basic
proteins) to form nucleosomes which, in turn,
orginize into chromosomes. During the course of
replication, the chromosomes are relaxed and the
nucleosomes get loosened.

DNA replication on the
lagging strand in
eukaryotes
(RPA – replication protein A; PCNA
– proliferating cell nuclear antigen;
RFC – eplication factor C; Rnase-H –
ribonuclease H; FENI – flap
endonuclease I
NOTE – leading strand not shown)

DNA repair by the base-excision repair pathway 25-23
1.DNA glycosilase recognize a
damage base and cleves between
the base and deoxyribose in the
backbone.
2.An AP endonuclease cleavesthe
phosphodiester backbone near the
APsite.
3.DNA polymerase I initiates repair
synthesis from the free 3 hydroxyl
at the nick, removing (with its 5 to
3 exonuclease activity) a portion of
the damaged strand and replaceing
it with undamaged DNA.
4.The nick remaining after DNA
polymerase I has dissociated is
sealed by DNAligase.

An example of error correction
by the 3'-5' exonuclease activity
of DNA polymerase
Structural analysis has located the
exonuclease activity ahead of the
polymerase activity as the enzyme in
oriented in its movement along the DNA.
A mismatched base (here, a C-Amismatch)
impedes translocation of DNA polymerase
I to next site. Sliding backward, the
enzyme corrects the mistake with its 3 to 5
exonuclease activiti, then resumes its
mistake with its 5 to 3 direction.

Nick translation
In this process, an RNA or DNA
strand paired to DNA template is
simultaneously degraded by 5 to 3
exonuclease activity of DNA
polymerase I and replaced by the
polymerase activity of the same
enzyme.These activities have a role
in both DNA repair and the
removal of RNA primers during
replication. Polymerase I extends
the nontemplate DNA strand and
moves the nick along the DNA – a
process called nick translation. A
nick remains where DNA
polumerase I dissociates, and later
seald another enzyme.

The cell cycle of a mammalia cell
(M – mitotic phase; G1 – Gap 1 phase; G0 – Gap 0 dormant phase; S phase – period of
replication; G2 – Gap 2 phase; )

Structure of RNA
RNA contains D-ribose.
RNA is a polymer of ribonucleotides held together by 3',5'-
phospodiester bridges.
RNA have specific differences
-the sugar in RNA is ribose (in DNAdeoxyribose)
-RNA contains pyrimidine uracil (in DNAthymin)
-RNA is usually a single-stranded polynucleotide
-Chargraff’s rule – non obey (due to single-stranded nature)
-alcali can hydrolyse RNA to 2',3'-cyclic diesters (it is
possible due to the presence of hydroxyl group at
2' position)
-RNA can be histologically identified by orcinolcolor
reaction due to presence of ribose.

Transcription
Transcription is a process in which ribonucleis acid is synthesised from
DNA. The word gene is refers to the functional unit of the DNA thatcan
be transcribed. The genetic information stored in DNA is expressed
through RNA. For this perpose, one of the two strands of DNA serves as
template (non-coding strand or antisense strand) – other DNA strand
which does not participate in transcription is reffered to as coding strand
or sense strand or non-template strand. Coding strand commonly used
since with the exception of T for U, promary mRNA contains codons
with the same base sequence.
The product formed in transcription is reffered to as primary transcript.
They undergo certain alterations (splicing, terminal additions, base
modification etc.) commonly known as post-transcriptional
modifications to produce functionally active RNAmolecules.

Transcription in prokaryotes
A single enzyme – DNA dependent RNA polymerase (or RNA
polymerase) synthesizes all the RNAs in prokaryotes – it is commonly
holoenzyme with five polypeptyde subunits - 2α, 1β, and 1β' and one
sigma factor.
Transcription involves three different stages^
Initiation
Elongation
Termination

Initiation
The binding of the enzyme RNA polymerase to DNA is a prerequisitefor
the transcription to start.
The specific region on the DNA where the enzyme binds is known as
promoter region. There are two base sequences on the codding DNA
strand which the sigma factor of RNA polumerase can recognise for
initiation of transcription.
1.Pribnow box (TATAbox) – consists of 6 nucleotide bases (TATAAT),
located on the left side about 10 bases away (upstream) from the starting
point of transcription.
2. The ‘–35’ sequence – is a second recognition site in the promoter
region of DNA – contains a base sequence TTGACA, which is locatewd
about 35 bases (upstream, hence –35) away on the left side from the site
of transcription start.

Elongation
As a holoenzyme, RNA polymerase recognise the promoter region, the
sigma factor is released and trancription proceeds. RNA is synthesisfron
5' end to 3' end antiparallel to the DNAtemplate.
For the formation of RNA – RNA polymerase utilizesribonucleotide
trophosphates (ATP, GTP, CTP and UTP)
The sequence of nucleotide base in the mRNA is complementary tothe
template DNAstrand.
RNA polymerase is differs from DNA polymerase in two aspects^
-no promer is required
-it does not possess endo- or exonuclease activity.
RNA polymerase has no ability to repaire the mistakes in the RNA
synthesized.

Termination
The process of transcription stops by termination signals. There are two
types of termination signals:
1.Rho (ρ) dependent termination (a specific protein, named ρ factor,
binds to the growing RNA [not to RNA polymerase] or weakly toDNA,
and in the bound state it acts as ATPase and terminates thetranscription)
2. Rho (ρ) independent termination – termination is brought about by the
formation of hairpins of newly synthesized RNA. This occures due to
the presence of palindromes – word that reads alike forward and
backward [madam, rotor]. As a result – the nwely synthesised RNAfolds
to form hairpins (due to complementary base pairing) that cause
termination of transcription.

Reaction of adding new nucleotide in 5' to 3' direction
(RNA polymerase).

Structure of the RNA polymerase

Transcription by RNA polymerase in E.coli (2)

Promoter region of DNA in prokaryotes

Synthesis of RNA from DNA template

Transcription in eukaryotes
Transcription in eucariotes, particulary termination is not clearlyknown.
RNA polymerases – there are three distinctone
RNA polymerase I – is responsible for the synthesis of precursorsfor
the large ribosomal RNAs
RNA polymerase II – synthesizesthe precursors for mRNA andsmall
nuclear RNAs
RNA polymerase III – participates in the formation of tRNA andsmall
ribosomal RNAs

Overview of transcriptin in eukaryotes

Promoter sites
In eukaryotes have a sequence of DNA bases which is almost identicalto
pribnow box of prokaryotes – known as Hogness box (or TATAbox)
located on the left about 25 nucleotides away (upstream) from the
starting site of mRNAsynthesis.
There also exists another site of recognotion between 70 and 80
nucleutides upstream from the start of transcription – is reffered as
CAAT box

Initiation of transcription
The molecular events required for the initiation of transcription involve
three stages:
1.Cromatin containing the promoter sequence made accessible to the
trancribtion machinery
2.Binding of transcription factors to DNA sequence in promoterregion
3.Stimulation of transcription by enhancers
Enhancer can increase gene expression by about 100 fold – this is made
possible by binding of enhancers to transcription factors to form
activators

Post-transcriptional modifications
Terminal base additions
Base modifications
Splicing
Different mRNA produced by alternate splicing
Messanger RNA
Transfere RNA
Ribosomal RNA

Translation
The genetic information stored in DNA is passed on to RNA (through
transcription) and ultimately expressed in the biosynthesis of protein or a
polypeptide in the living cell.

Genetic code used in translation
The three nucleotide (triplet) base sequence in mRNA that act aswords
for amino acids – codones

The genetic code along with respective amino acids

The Genetic code
The genetic code is:
Universal (the same codons are used to code for the same amino acid in
all the living organisms [however, there are a few exceptions])
Specific (a particular codon always codes for same amino acid)
Non-overlapping (read from a fixed point as a continuous base
sequence)
Degenerate (most amino acid have more than one codon)

DNA & RNA

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (9)

Similar to DNA & RNA

Similar to DNA & RNA (20)

More from Eneutron

More from Eneutron (20)

Recently uploaded

Recently uploaded (20)

DNA & RNA