Lecture 4: DNA transcription
1) What is the central dogma of molecular biology
2) What are the steps involved in transcribing a
primary RNA transcript?
3) How does eukaryotic post-transcriptional
processing convert a primary transcript into
messenger RNA?
4) Write notes on promoters, enhancers and
transcription factors
Central dogma of molecular
biology
Transcription
DNA directed RNA synthesis
What is the biological significance?
Allows selective expression of genes
Regulation of transcription controls time, place
and level of protein expression
Basic structure of a gene
Regulatory region
coding region
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E:\Lessons\5-4_Transc-Translb3\Transc-Transl.swf
Transc-Transl.htm
Transcription
Transcription is the mechanism by which a
template strand of DNA is utilized by specific
RNA polymerases to generate one of the
three different types of RNA.
Types of RNA
1) Messenger RNA (mRNA)
This class of RNAs are the genetic
coding templates used by the
translational machinery to
determine the order of amino acids
incorporated into an elongating
polypeptide in the process of
translation.
Types of RNA…..
2) Transfer RNA (tRNA)
This class of small RNAs form
covalent attachments to individual
amino acids and recognize the
encoded sequences of the mRNAs
to allow correct insertion of amino
acids into the elongating
polypeptide chain.
Types of RNA…..
3) Ribosomal RNA (rRNA)
This class of RNAs are assembled, together
with numerous ribosomal proteins, to form
the ribosomes. Ribosomes engage the
mRNAs and form a catalytic domain into
which the tRNAs enter with their attached
amino acids. The proteins of the ribosomes
catalyze all of the functions of polypeptide
synthesis
Where does transcription take place?
Transcription in eukaryotes
Step 1: transcribing a primary RNA transcript
Step 2: modification of this transcript into
mRNA
Step 1 - overview
A.Initiation
A) RNA polymerase
binds to promoter
& opens helix
B.Polymerisation
B) De novo synthesis
using rNTPs as
substrate
Chain elongation in
5’-3’ direction
C) stops at
C. Termination
termination signal
A) Initiation: ENZYME
RNA polymerase holoenzyme
an agglomeration of many different factors
that together direct the synthesis of mRNA
on a DNA template
Has a natural affinity for DNA
Initiation: SIGNAL
specific DNA sequences called promoters
1) Region where RNA polymerase binds to initiate
transcription
2) Sequence of promoter determines direction of RNA
polymerase action
3) Rate of gene transcription depends on rate of
formation of stable initiation complexes
PROMOTERS
Prokaryotes
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Near 5’ end of operons
Pribnow box – consensus sequence TATAAT
Fig 29-10: Voet and Voet
PROMOTERS
Eukaryotes
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Near 5’ end of genes
Recognised by RNA pol II
Consensus promoter sequence for
constitutive structural genes – GGGCGG
Selective structural genes – TATA
ENHANCERS
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Sequences that are associated with a promoter
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Enhance the activity of a promoter due to its
association with proteins called transcription
factors
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Enhancers mediate most selective gene expression
in eukaryotes
Polymerisation
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RNA polymerase binds to promoter & opens helix
RNA polymerase catalyses addition of rNTPs in the 5’-3’
direction
RNA polymerase generates hnRNAs (~70-1000 nt long) &
all other RNAs
Stops at termination signal
Termination
specific termination sequence
e.g E.coli needs 4-10A followed by a palindromic
GC rich region
Additional termination proteins
e.g. Rho factor in E.coli
Step 2: Modification
Post transcriptional processing
3 main steps
1) RNA capping,
2) polyadenylation
3) splicing
Post transcriptional processing
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Control of gene expression
following transcription but
before translation
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Conversion of primary
transcript into mature mRNA
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Occurs primarily in
eukaryotes
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Localised in nucleus
Post transcriptional
processing
1) Capping
Addition of 7
methylguanosine at 5’ end
Mediated by
guanylyltransferase
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Probably protects against
degradation
Serves as recognition site
for ribosomes
Transports hnRNA from
nucleus to cytoplasm
2) Tailing
Addition of poly(A) residues at 3’ end
 Transcript cleaved 15-20nt past AAUAAA
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Poly(A)polymerase and cleavage &
polyadenylation specificity factor (CPSF)
attach poly(A) generated from ATP
3) Splicing
Highly precise removal
of intron sequences
Performed by
spliceosomes (large
RNA-protein complex
made of small nuclear
ribonucleoproteins)
Recognise exon-intron
boundaries and splice
exons together by
transesterification
reactions
Cell type-specific splicing
Differential splicing in specific tissues
Regulation of gene
expression
Prokaryotes
Eukaryotes
• Other levels of regulation
• Mainly at
inlcude posttranscriptional
transcriptional level
and posttranslational
• Sets of genes
regulation
transcribed together
• Each gene transcribed
(polycistronic)
independently
(monocistronic)
• E.g. lac operon and trp
operon in bacteria
RNA polymerase
Prokaryotes
single multisubunit RNA polymerase
complex
RNA polymerase
Eukaryotes - 3 types exist
RNA pol I
RNA pol II
RNA pol III
Located in
nucleoli
Synthesises
most rRNA
precursors
Located in
nucleoplasm
Synthesises
mRNA
precursors
Located in
nucleoplasm
Synthesises 5S
rRNA, tRNA,
snRNAs
RNA polymerase
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Enzymes that catalyse the formation of RNA
using DNA as a template
De novo synthesis using rNTP as substrates
1960 – J Hurwitz & S Weiss
(RNA)n + rNTP = (RNA)n+1 + Ppi
Antibiotics such as Rifampicin / rifamycin B
inhibit RNA polymerase activity
Gene expression efficiency
When to
transcribe
gene?
How many
copies to be
transcribed?
DNA binding proteins
Proteins that recognise & bind to specific DNA sequences
Recognition determined by specific structural motifs
e.g. helix – loop –helix, zinc finger, leucine zipper
Examples include
Transcription factors
• general transcription factors
• Upstream transcription factors
• Inducible transcription factors
Activators
Repressors (silencers)
How does transcriptional control
differ in pro and eukaryotes?
Prokaryotes
Genes are usually switched ‘on’ by default
Repressor proteins needed to ‘stop’ transcription
Eukaryotes
Genes are usually switched ‘off’ by default
Transcriptional activators needed to induce transcription
Regulated by chromatin structure, DNA methylation etc
Lac operon
Fig 29-3/5
: Voet and Voet
Fig 8-20: Essential Cell Biology by Alberts et al