Topics 7, 9-10: DNA & Genomics
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. Double-stranded polynucleotide. Each strand forms a right-handed helic and 2 strands
coil to form a double helix
2. Diameter of helix is 2nm
3. Strands are antiparallel
4. Phosphate groups project outside of double helix while nitrogenous bases orientate
inwards toward the central axis at almost right angles to put the relatively
hydrophobic nitrogenous bases in the molecule’s interior, and thus away from the
surrounding aq medium
5. 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. histomes assemble into an octamer
30nm
700nm
• DNA is further coiled into 30nm chromatin fibre, aka solenoid
• Histone H1 and linker DNA is involved in this coiling
• nonhistone chromosomal proteins form a scaffold involved in
condensing the 30nm chromatin fibre to form looped domains
• in chromosomes, the looped domains fold and coil, further compacting
to produce the characteristic metaphase chromosome
*Particular genes always end up located at the same places in mitotic
and meiotic chromosomes => packing is highly specific and precise
Topics 7, 9-10: DNA & Genomics
Features
Genetic information is stored in the specific order of
base pairs
Relatively resistant to spontaneous mutations
Extensive H bonds
Hydrophobic interaction btw stacked base pairs
Only sugar phosphate backbone exposed to external
influences, nitrogenous bases tucked inside double helix
In eukaryotes, DNA is tightly wound around histones to
form repeating array of nucleosomes, that eventually
fold into higher order chromosomes
Specific, complementary base pairing between DNA
strands
Benefits
Base sequence is stable and invariant
Stable storage of genetic information
Stabilise the DNA double helix
Prevent DNA from thermal and
physical damage, and facilitate their
segregation onto daughter nuclei
Genetic information is redundant, cell
is able to discard the damaged strand
of the two, and use the remaining as
template -> maintain DNA’s integrity
Replication of DNA
Semiconservative model: When a ds-DNA replicates, each of the 2 daughter DNA molecules
has one old strand derived from parental DNA, and one newly made strand
2 strands of
DNA are
complementary
A. Location of origins
of replication ->
formation of
replication bubble
F. Daughter DNA
molecule has one
old and one new
strand
1 of the strands serves
as a template, forming
a new complementary
daughter strand
there are now 2 ds-DNA, each an exact
replica of the parental DNA to ensure
faithful transmission of genetic
information
B. Separation of
parental DNA
strands by helicase
and unwinding by
topoisomerase
C. Synthesis of RNA
primers for DNA
polymerase to
initiate synthesis
E. Simultaneous
synthesis of leading
and lagging strands
D. Synthesis of
daugther DNA
strands via
complementary
base pairing
*Origin of replication usually has a stretch of A-T rich sequence as only 2 H bonds are formed
btw A-T base-pair, as compared to 3H bonds btw C-G
Topics 7, 9-10: DNA & Genomics
A. 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. Al each end of a replication
bubble is a replication fork, a Y-shaped region where the new strands of DNA are elongating.
Replication of DNA then proceeds in both directions in 5’ to 3’ direction. Multiple replication
bubbles may form and eventually fuse, thus speeding up the copying of very long DNA
molecules.
B. Separation of parental DNA strands by helicase and unwinding by topoisomerase
DNA double helix is unwound, facilitated by topoisomerase, which nicks a single strand of DNA and
creates a transient break. This relieves stress on the DNA molecule by allowing free rotation
around the single strand. This may be required to help unwind the double helix ahead of the
replication fork. Helicase then separates the parental DNA strands, using ATP as source of energy,
to break H bonds and keeps strands apart. Replication fork stability is maintained by single-strandbinding proteins (SSB) which bind to single-stranded regions of DNA tightly to keep ssDNA from
being degraded, straighten out the ssDNA template, and prevent it from reannealing. This ensures
that the DNA is still readable by DNA polymerase.
C. 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. DNA polymerase I later replaces the RNA nucleotides of
the primers with DNA versions.
D. Synthesis of daughter DNA strands via complementary base pairing
The parental DNA strands, separated at the replication fork and primed with a RNA primer on each
strand, form the templates along which deoxyribonucleoside triphosphate (dNTPs) align
themselves. The alignment of dNTPS to the growing daughter DNA stand is determined by
complementary base pairing. With an RNA primer anchoring the start of the daughter DNA strand,
DNA polymerases carry out the polymerisation of the strand by catalysing the formation of
phosphoester bond between 3’ OH group of a growing daughter DNA strand and an incoming
nucleotide. Due to the active site specificity of DNA polymerases, synthesis of both daughter DNA
strands can only occur in the 5’ to 3’ direction.
E. Simultaneous synthesis of leading and lagging strands
Leading and lagging strand synthesis are concurrent. Lagging strand is discontinuously synthesised
as a series of Okazaki fragments, polymerised against the overall direction of the replication fork.
Each Okazaki fragment requires an RNA primer for strand initiation. The Okazaki fragments are
then ligated to produce a continuous DNA strand. DNA polymerase I removes the RNA primer and
replaces it with dNTPs. DNA ligase then catalyses the formation of a covalent bond between the 3’
end of each new Okazaki fragment and the 5’ end of the growing daughter strand.
Topics 7, 9-10: DNA & Genomics
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.
Central Dogma of Molecular Biology: The principle of directional informational flow from DNA to
RNA to protein.
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.
Translation: The process, in which a polypeptide chain is synthesised by ribosomes using genetic
information encoded in an mRNA template
DNA → pre-mRNA → mature mRNA
tRNA & rRNA
polypeptide
Gene: A section of the DNA that contains the information in the form of a specific sequence of
nucleotides to direct the synthesis of one polypeptide chain or RNA. It is a unit of inheritance
located in the locus on the chromosome which specifies a particular character of an organism.
Characteristic RNA
DNA
Substituent
Made of polynucleotides, basic units: phosphate group, pentose sugar,
nitrogenous base
Bond
Have a sugar-phosphate backbone joined by phosphodiester bonds
Synthesis
Polymerised through condensation synthesis
Synthesised by complementary base pairing of nucleotides using a template
Nitrogenous A, G, U, C
A, G, T, C
bases
Size & mass
Smaller molecular mass (20k to 2000k
Larger molecular mass (100k to
Da)
150000k Da)
No. 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.
complex tertiary structure eg. 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
Topics 7, 9-10: DNA & Genomics
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. Each codon within the coding region
represents an aa in the corresponding aa sequence in the protein
Serves as an adaptor molecule in protein synthesis. 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
Topics 7, 9-10: DNA & Genomics
RNA polymerase I transcribes the genes encoding rRNA.
RNA polymerase II transcribes most genes, including all those that encode proteins and mRNA.
RNA polymerase III transcribes the genes encoding tRNA. All 3 polymerases are found in
nucleoplasm.
General/basal transcription factors are required to:
1. position RNA polymerase correctly at the promoter
2. separate 2 strands of DNA to allow transcription to begin
3. release RNA polymerase from the promoter to begin elongating the RNA against the DNA
template once transcription has begun
Transcription
Initiation: RNA polymerase binds to promoter
Elongation: polymerase moves downstream, elongating RNA transcript 5'→ 3'
Termination: RNA transcript is released and polymerase detaches from DNA
Stage
Initiation
Elongation
Step
Formation of
Transcription
Initiation
Complex
Process
 A collection of proteins called general transcription factors are
assembled along the promoter
 TFIID binds to the TATA box, found within the promoter
 General transcription factors mediate the binding of RNA
polymerase to the promoter, forming the transcription
initiation complex
Unwinding of
 Binding of RNA polymerase to the promoter causes the DNA
DNA helix &
double helix to unwind and the 2 strands separate
separation of
 During which, H bonds btw complementary pairs are disrupted,
the 2 strands
creating a transcription bubble which exposes a short stretch of
nucleotides on each strand
Assembly of
 One of the 2 exposed DNA strands act as a template for
ribonucleotides
complementary base pairing to direct the assembly of incoming
and formation
NTPs
of 1st phospho-  RNA polymerase catalyses the first phosphodiester bond
diester bond
 This marks the end of initiation
Movement of
 As RNA polymerase moves along the template DNA in the 3’ to
transcription
5’ direction, DNA double helix continues to transiently unwind
bubble
Elongation of
 NTPs are added through complementary base pairing with the
polynucleotide
DNA template
 As each 5’ end of NTPs are brought in, its 2 terminal phosphates
Topics 7, 9-10: DNA & Genomics
are removed and the remaining group is added to the free 3’
OH group of the growing RNA chain via phosphodiester bond
Re-annealing
 RNA polymerase reanneals the unwound DNA behind it,
of DNA and
dissociating the growing RNA chain from the template
proofreading
 It carries out proofreading functions and is responsible for the
removal of incorrectly inserted ribonucleotides
Termination
 RNA polymerase transcribes a terminator sequence in the DNA
 Triggers the release of the RNA chain and dissociation of the
RNA polymerase from the DNA
 Transcribed terminator codes for a polyadenylation sequence
(AAUAAA)
Exons: protein-coding sequence in the gene
Introns: long sequences of nucleotides inserted between exons that do not code for any portion of
the polypeptide, ie. 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. Catalysed by
poly(A)-polymerase
RNA splicing
RNA splicing occurs after the
 Provides for variation as different
release of pre-mRNA from RNA
combination of exons could results in
polymerase, during which, introns
different types of polypeptides
are removed while remaining
synthesised
exons are spliced/ligated together
to form mature mRNA. 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
Topics 7, 9-10: DNA & Genomics









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.e. 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. The first amino acid of a polypeptide chain, and the reading frame
used from that point on
Stop codon: UAA, UAG and UGA are stop signals marking the end of protein synthesis. They do not
code for any amino acid. There is no tRNA with an anticodon complementary to these 3 codons.
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
Topics 7, 9-10: DNA & Genomics
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
Topics 7, 9-10: DNA & Genomics
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 a biochemical functional group, such as acetate, methyl, phosphate, various
lipids and carbohydrates
2. 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. Making structural changes, like the formation of disulfide linkages
4. Removing a sequence of amino acids, or cutting the peptide chain in the middle
5. Folding the polypeptide into a specific 3D conformation
Control of Eukaryotic Gene


Amount (quantity), types (quality) of gene products
Timing of appearance of certain gene products
Gene Amplification: production of multiple copies of a specific gene to amplify the quantity of the
gene product. 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. These minichromosomes accumulate within the nucleus of
the oocyte and serve as DNA templates for transcription of rRNA genes to produce rRNA
Significance: this increase in transcription of rRNA genes due to large amount of templates
available is necessary to accommodate the enormous amount of ribosome biosynthesis that must
take place during oogenesis which is in turn required to sustain the high rate of protein synthesis
needed for early embryonic development upon fertilisation. This amplification phenomenon is
developmentally regulated, since it occurs only during the development of the oocyte.
Topics 7, 9-10: DNA & Genomics
Transcriptional Control


Gene accessibility (histone acetylation and DNA methylation)
Initiation of transcription
Gene Accessibility
Process
Mechanism
Enzymes
Site
Outcome
DNA methylation
Histone modification
Deacetylation
Acetylation
 Cytosine (C) nucleotides in the  Histone deacetylases  Positively-charged lysine
sequence 5’-CG-3’ (CpG
(HDACs) catalyse the
residues in the histone tails
dinucleotides) can be
deacetylation of
can be acetylated by histone
methylated by DNA methylacetylated lysine
acetyl transferase (HATs)
transferase to add a methyl
residues in histone
 Positive charge on lysine
group
tails
residues is neutralised and
 Methylation within the
 Lysine residues
becomes uncharged
promoter prevents
become positively Reduction in affinity of the
transcription
charged again,
histone complex for the
resulting in an
 Change in 3D conformation of
DNA molecule
increase in affinity of  Chromatin structure
DNA prevents binding of
the histone complex
transcription factors to
becomes less compact,
for the DNA molecule
promoter
exposing DNA regions of
 Methylated DNA serve as
genes to transcription
recognition signals for methyfactors and RNA
CpG-binding proteins (MeCPs)
polymerases
that in turn recruit other
proteins such as HDACs to
modify chromatin structure
such that it becomes more
condensed
DNA methyl transferase
HDACs
HATs
CpG islands
Histone tails
Down regulate transcription
Down regulate
Up regulate transcription
transcription
Gene & Intergenic DNA
Gene
Regulatory sequences
are promoter and
regions found in 5’ and
3’ UTR
Introns may also contain
regulatory sequence
Intergenic
Regulatory sequences,
telomeres, centromeres
and origin of replication
Regulatory sequences (control
elements)
Promoter
Includes TATA box, which resides app
25-30 bp upstream of transcription start
site
RNA polymerase and general
transcription factors assemble to form
transcription initiation complex
Promoter-proximal
Found within 100-200 bp upstream of
transcription start site
Essential for maximum rate of
transcription
Binding sites for general transcription
Specific transcription
factors
Activator
Bind to enhancers,
triggering a series of
interaction that results in
an increased rate of
transcription
Repressor
Bind to silencers, triggering
a series of interaction that
results in a decreased rate
of transcription
Topics 7, 9-10: DNA & Genomics
factors
Distal
Include enhancers and silencers
Can greatly increase/ decrease rate of
transcription rates
Act at a distance of >200-1000s of bp
upstream from the promoter or
downstream from the final exon of a
gene
May be within an intron
Action of Enhancers and Activators leading to initiation of transcription:
1. Activators bind to their respective enhancers
2. DNA-bending protein causes the looping of DNA which allows activators bound to
enhancers that are lovated far away to tbe brought close to the promoter
3. Upon binding to enhancers, the activator interacts with components of the transcription
machinery including, general transcription factors and RNA polymerase
4. This results in the improved recruitment of general transcription factors and RNA
polymerase to the promoter and their interaction with the promoter to form a
transcription initiation complex
5. Activator also helps proper positioning of the transcription initiation complex on the
promoter to initiate transcription. The rate of transcription is increased.
6. Some activators that are unable to interact directly with RNA polymerase and general
transcription factors interact with mediator proteins, that serve as adaptor molecules to
help integrate signals from activators
7. 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. In eukaryotes, the precise control of transcription depends largely on the binding of
activators to their respective enhancers. 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
Topics 7, 9-10: DNA & Genomics
Splicing of mRNA:
1. the cleavage at the 5’ splice site and joining of the intron to a branch point within the
intron
2. this reaction yields a lariat-like intermediate, in which intron forms a loop
3. the cleavage at the 3’ splice site and simultaneous ligation of the exons, resulting in an
exision of the intron as a lariat-like structure
4. 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