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DNA Structure & Function
5’ end

Phosphate group
(phosphoester bond at 5’Carbon)
Pentose sugar
(glycosidic bond at 3’ Carbon)
Nitrogenous base

3’ end
Purine X Pyrimidine
A
C

T/U
G

pentose + nitrogenous base = nucleoside
nucleoside + phosphoric acid = nucleotide
(nucleotide)n = nucleic acid

DNA STRUCTURE:
1
...
Each strand forms a right-handed helic and 2 strands
coil to form a double helix
2
...
Strands are antiparallel
4
...
Opposite strands are connected by weak H bonds
• DNA is packed into nucleosomes to produce 10nm chromatin fibre
• Histones proteins, highly concentrated in positively-charged residues,
form ionic bonds with negatively-charged sugar phosphate backbone
10nm
• -> nucleosome core
...
Location of origins
of replication ->
formation of
replication bubble

F
...
Separation of
parental DNA
strands by helicase
and unwinding by
topoisomerase

C
...
Simultaneous
synthesis of leading
and lagging strands

D
...
Location of origins of replication -> formation of replication bubble
Proteins that initiate DNA replication recognise this sequence and attach to the DNA,
separating the two strands and opening up a replication bubble
...

Replication of DNA then proceeds in both directions in 5’ to 3’ direction
...

B
...
This relieves stress on the DNA molecule by allowing free rotation
around the single strand
...
Helicase then separates the parental DNA strands, using ATP as source of energy,
to break H bonds and keeps strands apart
...
This ensures
that the DNA is still readable by DNA polymerase
...
Synthesis of RNA primers for DNA polymerase to initiate synthesis
A portion of the parental DNA strand serves as a complementary base sequence for primase to
join ribonucleotides to make the primer
...

D
...
The alignment of dNTPS to the growing daughter DNA stand is determined by
complementary base pairing
...
Due to the active site specificity of DNA polymerases, synthesis of both daughter DNA
strands can only occur in the 5’ to 3’ direction
...
Simultaneous synthesis of leading and lagging strands
Leading and lagging strand synthesis are concurrent
...

Each Okazaki fragment requires an RNA primer for strand initiation
...
DNA polymerase I removes the RNA primer and
replaces it with dNTPs
...


Eukaryotic Gene Expression
Gene Expression: The process in which the information within a gene is used, first to synthesise
RNA, through transcription, and then to a protein, through translation, eventually to affect the
phenotype of an organism
...

Transcription: The process, in which a complementary RNA copy is made under the direction of
the template strand of a specific region of the DNA molecule, catalysed by the enzyme RNA
polymerase
...
It is a unit of inheritance
located in the locus on the chromosome which specifies a particular character of an organism
...
of
1 polynucleotide chain
2 polynucleotide chains
subunits
3D structure Almost always single-stranded, helical
Always a double-stranded helical
molecule, which can be folded into a
molecule which forms a double helix
...
tRNA
Coiled around histone proteins
Monomers
Ribonucleotides
Deoxyribonucleotides
Pentose
OH group attached on 2’Carbon
H attached on 2’ Carbon
sugar
Chemical
Less stable – more reactive partly due
More stable – more resistant to
stability
to ribose having an additional reactive spontaneous enzymatic breakdown
2’ OH group
due to deoxyribose lacking 2’OH group
Purines :
A: U ≠ G:C ≠ 1:1 (Ratio cannot be
A:T = G:C = 1:1
pyrimidines
predicted as RNA is single-stranded,
(Chargaff’s rule)
without a complementary strand)
Basic forms
Several basic forms: messenger RNA,
Only one basic form
transfer RNA, ribosomal RNA, small
nuclear RNA, small interfering RNA

Location

Synthesised in the nucleus but found
throughout the cell

Amount/cell

Amount varies from cell to cell

Types of RNA
Messenger RNA (mRNA)

Transfer RNA (tRNA)

Ribosomal RNA (rRNA)
Small nuclear RNA
(snRNA)
Small interfering RNA
(siRNA) & micro RNA
(miRNA)

Found almost exclusively in the
nucleus with exception of
mitochondria and chloroplast
Amount is constant for all somatic
cells in a species

Functions
Serves as an intermediate that carries information from DNA, acting
as a template for translation
...
Used to bring in
specific aa in a sequence corresponding to the sequence of codons
in mRNA
Plays catalytic and structural roles in ribosomes
Plays structural and catalytic roles in spliceosomes, the complexes of
protein and RNA which carry out splicing of pre-mRNA
Involved in regulation of gene expression
miRNA prevents gene expression either by degrading the target
mRNA or by blocking its translation

Promoter
Structure  Contains RNA
polymerase
binding site &
transcription
start site
 Contains a TATA
box
 Promoter is not
transcribed
except for the
nucleotides after
the start site
Function  TATA box serves
as a binding site
for a general
transcription
factor called
TFIID
 subsequently
facilitating the
binding of RNA
polymerases
 determines
which of the 2
strands of DNA is
used as template

Coding region
Terminator
RNA polymerase
 Segment of DNA that  Found at the  Enzyme comprising of
is transcribed into a
end of a
several protein subunits
single-stranded
gene
and is found in the
mature mRNA
nucleoplasm
 Whole
 Flanked by promoter
terminator is  Simultaneous
and terminator
transcribed
transcription from same
DNA template is possible
as namy RNA
polymerases can be
transcribing different
parts of the same gene
simultaneously
 only 1 of the 2
 codes for a  responsible for the
strands serves as the
polyadenyla- synthesis of RNA using
template for
tion signal
ribonucleoside
transcription
sequence
triphosphate (NTP), in 5’
(AAUAAA) in → 3’
 read in 3’ to 5’
direction to facilitate the pre catalyse the assembly of
mRNA/
synthesis of RNA in
ribonucleotides and the
primary
5’ to 3’ direction
formation of
transcript
phosphodiester bond
 template strand
btw free 5’ phosphate
serves a template to  terminates
transcription group of incoming NTP
direct synthesis of
and free 3’ OH group of
RNA molecule by
growing RNA
complementary base
polynucleotide chain
pairing

RNA polymerase I transcribes the genes encoding rRNA
...

RNA polymerase III transcribes the genes encoding tRNA
...

General/basal transcription factors are required to:
1
...
separate 2 strands of DNA to allow transcription to begin
3
...
are non-coding sequences
Post-transcriptional Modification
Modification
Process
Function
Addtion of 5’
The 5’ end of the new pre-mRNA
 Protects mRNA from degradation by
Methylguanosine molecule is modified by addition
nucleases and phosphatises that
cap
of a cap that consists of a
degrade the RNA from the 5’end during
methylated guanine (G)
its transport from the nucleus to the
nucleotide/ methylguanosine
cytoplasm
triphosphate
 5’ cap signals the 5’ end of the mRNA
which serves as the assembly point to
recruit the small subunit of the
ribosome for translation to begin
 Helps distinguish mRNAs from the
other types of RNA molecules
Addition of 3’
Immediately after the pre-mRNA is  3’ poly(A) tail protects the mRNA from
poly(A) tail
cleaved by an endonuclease at a
degradation by nucleases
site 10-35 nucleotides after the
 Make mRNA a more stable template for
AAUAAA poly(A) sequence, the 3’
translation
end of the pre-mRNA is modified
 Required to facilitate the export of
by addition of a series of ~200
mRNA out of the nucleus via nuclear
adenine (A) nucleotides, referred
pores
to as the poly(A) tail
...
Requires
hydrolysis of ATP
Genetic code:



consists of information in the form of 3 nucleotide bases called codons of mRNA
also the triplet bases in the non-template/non-transcribed strand of DNA











of the 64 possible codons, 61 code for amino acids, including the start codon (AUG), while
3 serve as termination signals of polypeptide synthesis, i
...
stop codons (UAG, UAA, UGA)
is a triplet code- each mRNA codon that specifies an amino acid in a polypeptide chain
consists of 3 nucleotide bases
linear code- always read in the 5’ to 3’ direction
almost universal- same code is used by all organisms
continuous and non-overlapping- nucleotides in mRNA are read continuously, as
successive groups of 3 nucleotides, one codon at a time without skipping any nucleotides
degenerate, but unambiguous- a single amino acid can be coded by >1 different codon,
but every codon codes for just one amino acid
Degenerate codons differ only in the 3rd position of the codon
Wobble base phenomenon- a single tRNA can recognise 2 or more of these degenerate
codons
Has punctuation codons – start and stop codons

Start codon: start signal for protein synthesis is the start codon AUG, which codes for the
incorporation of methionine
...
They do not
code for any amino acid
...

Translation
Amino acid activation by aminoacyl-tRNA synthetase
Initiation: mRNA, initiator tRNAMet, and the 2 subunits of a ribosome are brought together
Elongation & translocation: amino acids are added to the growing polypeptide chain from N to C
terminal
Termination: stop codon in mRNA reaches A site of ribosome
Stage
Preparation

Step
Amino acid
activation by
aminoacyltRNA
synthetase

Process
 Aminoacyl-tRNA synthetases recognise the specific anticodon
sequence on a tRNA molecule as well as the specific amino acid
 Each of the 20 different amino-acyl-tRNA synthetases covalently
attach a specific amino acid to the 3’CCA stem of its appropriate
set of tRNA molecules via an ester linkage, forming aminoacyltRNA, aka activated amino acid
 Hydrolysis of ATP is required
 Active site of each aminoacyl-tRNA synthetase must be
complementary to the conformation of the specific amino acid
and specific anticodon sequence of the tRNA in order for them
to bind

Binding of
 Eukaryotic initiation factor (eIFs) bind to the small 40s subunit
initiation
of a ribosome and position the initiator tRNAiMet which carries a
factors to small
methionine to its P site
subunit
Binding of
 The small subunit then binds to the mRNA by recognition of its
small subunit
5’ methylguanosine cap
to mRNA
 The small ribosomal subunit then moves downstream in the 5’
to 3’ direction along the mRNA in search of the start codon AUG,
which signals the start site of translation
Association of  The tRNAiMet associates with the start codon on the mRNA
tRNAiMet &
through complementary base pairing
formation of
 The tRNAiMet has a unique anti-codon loop that is distinct from
initiation
that of the tRNA that normally carries methionine
complex
 This is followed by the dissociation of eLFs which allows for the
binding of the large 60S ribosomal subunit, completing an 80S
translation initiation complex
 The tRNAiMet sits in the P site of the ribosom, and the initial
methionine forms the N-terminus of the polypeptide
 The A-site is vacant, waiting for entry of the next aminoacyltRNA complementary to the second codon of the mRNA
Elongation & Codon
 After the formation of initiation complex, an aminoacyl-tRNA
translocation recognition
carrying the 2nd amino acid in the chain binds to the ribosomal A
and aminoacylsite via complementary base pairing between its anticodon and
tRNA binding
the codon in the mRNA exposed at the A site and is held in place
by H bonds
 tRNAs are brought in by elongation factors with the hydrolysis of
GTP as an energy source
Peptide bond
 When the 2nd tRNA is bound to the ribosome, its amino acid is
formation
placed directly adjacent to the tRNAiMet
 Peptidyl transferase in the large ribosomal subunit catalyses the
formation of a peptide cond between the carboxyl end of
methionine and the amino group of the 2nd amino acid
 The methionine is thus transferred to the 2nd amino acid carried
by the aminoacyl-tRNA at the A site
 The ester bond between the initial methionine and its tRNA is
broken to release the initial methionine
 The deacylated tRNA lies in the P site, while the new peptidyltRNA has been created in the A site
Translocation
 The ribosome is traslocated one codon in the 5’ to 3’ direction,
guided by elongation factors, with the hydrolysis of GTP
 This relocates the initial deacylated tRNA to the E site from
where it diffuses out of the ribosome
 Repositions the peptidyl-tRNA at the P site and exposes the
next codon on the mRNA at the A site
Termination stop codon in
 Termination occurs when a stop codon in the mRNA reaches
mRNA reaches
the A site of the ribosom
A site of
 A release factor binds directly to the stop codon at the A site
ribosome
 The release factor causes the addition of a water molecule
Initiation

instead of an amino acid to the polypeptide chain
 This frees the carboxyl end of the completed polypeptide from
the tRNA in the P site by hydrolysis
 Polypeptide is released through the exit tunnel of the ribosomal
large subunit
 Ribosome then releases the mRNA and separates into large and
small subunits
 tRNA molecules may then be recycled and used to form new
aminoacyl-tRNAs
Post-translational Modification:
1
...
Attaching to ubiquitins, which marks proteins for proteolysis by proteasomes, allowing for
the control of the length of time in which a protein can function
3
...
Removing a sequence of amino acids, or cutting the peptide chain in the middle
5
...
This increase in the copy number of gene is a result of repeated rounds of DNA
replication at a particular chromosomal region
Example: Ribosomal RNA gene amplification in the frog Xenopus laevis
Observation: During the development of the oocyte in the frog Xenopus laevis, the original 500
copids of genes that encode for rRNA genes are amplified through repeated rounds of replication
to about 4000-fold, so that the mature oocyte contains about 2 million copies of the genes for
rRNA
Process: many copies of circular DNA molecules called minichromosomes each containing 1 to 20
copies of the rRNA genes are formed
...
This amplification phenomenon is
developmentally regulated, since it occurs only during the development of the oocyte
...
Activators bind to their respective enhancers
2
...
Upon binding to enhancers, the activator interacts with components of the transcription
machinery including, general transcription factors and RNA polymerase
4
...
Activator also helps proper positioning of the transcription initiation complex on the
promoter to initiate transcription
...

6
...
Activators bind to the mediator proteins, and this facilitates the correct positioning of GTF
and RNA polymerase at the promoter, allowing for the formation of the transcription
initiation complex
8
...
The particular combination of enhancers
associated with a gene will be able to activate transcription only when the appropriate
activators are present during precise timing of development or in a specific cell type like
liver(albumin)/lens(crystallin) cell

5’cap
Addition of 7methylguanosine
triphosphate

Post-transcriptional Modifications
3’ poly(A) tail
RNA splicing
Alternative splicing
Polyadenylation
Removal of
Use of different splicing sites
resulting in 3’
introns while the resulting in alternative splicing, which
poly(A) tail about
remain exons are allows different exons to be joined
200 nucleotides
ligated together
together in different combinations
long
by spliceosome at This produces from the same primary
splice sites
transcript, different mRNAs which in
turn generate different proteins

Splicing of mRNA:
1
...
this reaction yields a lariat-like intermediate, in which intron forms a loop
3
...
DNA sequence at the 5’ and 3’ ends of an intron serve as recognition sites for spliceosomes
to bind

Stability of mRNA

Determines the
duration for which
translation can occur
Rate of degradation
determined by
sequences in 3’UTR
Path2:Internal
cleavage of mRNA:
an endonuclease
cleaves the mRNA
internally and poly(A)
tail is removed in 1
step→ path 1
Path 1: poly(A) tail
shortening:
Poly(A) tail is
shortened to critical
length by
exonuclease
5’ cap is removed and
exposed mRNA is
rapidly degraded
from 5’ end
At the same time,
mRNA continues to
be degraded from 3’
end

Translationary Control
Initiation
Alternative
translational
initiation sites
Eukaryotic Initiation Use of 2nd or
Factors: Initiation of subsequent AUG for
translation is
translation initiation:
dependent upon a
sometimes the
host of translation
scanning small
initiation factors:
ribosomal subunit
eukaryotic initiation skips the first AUG
factors
codon and uses the
By varying the
2nd or subsequent
abundance and
AUG to initiate
activity of these
translation – “leaky
factors, it is possible scanning”
to affect the rate of Results in proteins
translational
that vary in their
initiation
amino-terminal
sequence
Translational
Initiation of
Repressors: bind to translation in the
various regions of
middle of mRNA: an
the mRNA, usually
internal ribosome
the 5’ or 3’ UTRs,
entry site (IRES) is a
and interfere with
specialised nucleotide
the initiation of
sequence that allows
translation by
for translation
blocking the
initiation in the
attachment of
middle of a mRNA
ribosomes or other
sequence in a captranslation initiation independent manner
factors
Protein with different
primary structure is
produced

RNA interference

Micro-RNA
(miRNA)encoding
genes are transcribed,
synthesising RNA
transcripts that fold
back on themselves,
forming a hairpin
structure, held
together by H bonds
They are then
proceddes by Dicer,
cutting the dsRNA
transcripts into
smaller fragments
One strand is
degraded by TNAinducing silencing
complex (RISC), while
the remaining strand
binds to RISC to form
miRNA-protein
complex that bind to
mRNA molecules with
complementary
sequences, thus
inhibiting
translation/degrading
mRNA



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