The genetic code & gene

expression

Basis of the genetic code

Genes and Transcription

Christine Carrington
Oct 2020
 What is a gene?

• The basic physical and functional unit of heredity.

• A collection of adjacent nucleotides in DNA that specify the amino
acid sequence of a polypeptide chain.
• A sequence of nucleotides in DNA or RNA that encodes the
synthesis of a gene product, either RNA or protein.
 Gene Expression
(protein coding genes)

DNA
TRANSCRIPTION (T/C) : the
synthesis of RNA from a DNA
tRNA, rRNA template

mRNA
TRANSLATION: the process by
which the genetic code carried on
mRNA is used in the synthesis
of a protein

POLYPEPTIDE
 The Genetic Code
• The genetic information in DNA is solely in the sequence of the
four nitrogenous bases A, T, G, C (A, U, G, C in mRNA).
• The code is triplet, non-overlapping, degenerate, universal.

Partial nucleotide SARS CoV-2 genome sequences aligned with Wuhan-Hu-1 reference sequence
 The code is triplet

• There are 20 aa and only 4

bases
• Minimum no. of bases which
together can encode 20 aa is
three
• 3 adjacent bases =1 aa
• These three adjacent bases
are called codons

NHGRI http://www.genome.gov/page.cfm?pageID=10000552 by artist Darryl Leja

 The code is
non-overlapping
Each nucleotide
belongs to only one
codon

NHGRI http://www.genome.gov/page.cfm?pageID=10000552 by artist Darryl Leja

The code is degenerate
 ost
a lm
The code is universal
• Vast majority of genes use same genetic code.
• Rules (i.e. triplet, non-overlapping) are the same
• Some slight variations in terms of what amino acids specific codons encode.
 os t
a lm
The code is universal

Various alternative
GUG can be used
mitochondrial codes
as start codon in
bacteria and
UUG can be used
archaebacteria
as start codon in
In mycoplasma UGA =
bacteria Tryptophan
and instead of
archaebacteria STOP
CUG = Ser instead
of Leu in yeasts of
”CTG clade” e.g.
Candida albicans
 os t
a lm
The code is universal

Translational readthrough may

occur in some genes: stop codons
occasionally used to encode amino
acids
 Gene Expression
(protein coding genes)

• Step 1: Transcription
– synthesis of an RNA copy of the gene
– in eukaryotes this is followed by post transcriptional modification of the
primary RNA transcript

• Step 2 : Movement of mRNA from nucleus to cytoplasm

• Step 3: Translation
– Synthesis of new polypeptide using mRNA as a template
– Post-translational modification of polypeptide may occur.
 Structure of a eukaryotic gene
Enhancer/
silencer -40
Other
sequences sequence -25 sequence introns
regulatory
elements

Elements Promoter
under - site of RNA pol binding exon
hormonal -sequence determines/
control controls basal level of Structural gene
expression
 Gene Expression
• Transcription is the synthesis of RNA on a DNA template

DNA template + nNTP

RNA polymerase

DNA template + RNA + (n-1)PPi

Note: In contrast to DNA polymerase, RNA polymerase

does not require a primer
 Mechanism of transcription
n All bacterial and eukaryotic RNA is synthesised from
a DNA template by a DNA dependent RNA
polymerases.
 RNA polymerases

• RNA polymerases are

multi-subunit enzymes

• E. coli enzyme:
– Five subunits (2a, 2b and s)
– 2a and 2b = core enzyme
(responsible for transcription;
2a, 2b and s = holoenzyme
– s subunit required for promoter
recognition & initiation
 RNA polymerases (cont’)

• Eukaryotes possess three types of RNA pol:

– RNA pol I makes rRNA
– RNA pol II makes mRNA
– RNA pol III makes tRNA, 5s RNA & snRNA
 What is a promoter?
• Promoter is the site which RNA pol recognises
at the beginning of a transcribed region.

Transcribed region?

In eukaryotes transcribed region = a gene

(region encoding one polypeptide)

In prokaryotes and viruses, several genes may be

transcribed at once à several polypeptides encoded on a
single RNA transcript.
 Transcription divided into three parts:

• Initiation
– Recognition of active gene starting point (promoter) by RNA
polymerase and beginning of bond formation
• Elongation
– Synthesis of RNA chain
• Termination & release
– End of synthesis and release of newly synthesized RNA from
template DNA
 Transcription - Initiation
• Several transcription factors bind to
promoter.

• RNA pol (including s factor) binds to the

promoter to form a closed complex
– Orientation and frequency of binding
determined by the s factor
– Different s factors recognise different
promoters

• RNA pol becomes more tightly

associated; local unwinding of 10bp
(open complex)

• Initiating NTP binds and phosphodiester

bond formed with next NTP

• s factor released
 Transcription - Elongation
• NTPs bind to RNA pol
and form hydrogen
bonds with the template.

• There is bond formation

between the new NTP
and the nucleotide at the
3’ end of growing chain.

• The RNA pol

translocates so active
site is free to bind new
NTP.
Termination
Prokaryotes
• Rho independent termination
sequence at region of termination such that
there is formation of a stem-loop-stem
structure which leads to termination

• Rho dependent termination

dependent on a rho factor which has RNA
dependent ATPase activity

Eukaryotes
terminator signals are recognized by protein
factors associated with RNA polymerase II
and trigger the termination process
Fate of RNA transcript in prokaryotes versus eukaryotes
Eukaryotes Prokaryotes

RNA
processing

©1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts,

Walter. http://www.garlandscience.com/ECB/about.html
Published by Garland Publishing, a member of the Taylor & Francis Group.
 5’ cap on eukaryotic mRNA
The cap consists of a methylated guanosine molecule added to the 5’
end on the RNA

Other nucleotides
may also be
methylated
 Splicing

• Introns are
removed
and exons
rejoined to
form
complete
coding
sequence
 Poly A tail on eukaryotic mRNA

• Stretch of A residues at
the 3’ end of the mRNA.

• Confers stability on the

mRNA.

• NOT encoded in DNA

Prokaryotic mRNAs can encode more than one polypeptide
 Transcriptional Control
Enhancer/
silencer -40
Other
sequences sequence -25 sequence introns
regulatory
elements

Elements Promoter
under - site of RNA pol binding exon
hormonal -sequence determines/
control controls basal level of Structural gene
expression
 Transcriptional control
introns

Polymerase II
complex +1
TATA

Cis-regulatory Element exons

Core Promoter
Structural gene
• Core promoter
– Binding site for basal transcription complex (polymerase II complex)
• RNA polymerase II
• numerous transcription factors
• TATA binding protein
– Contains TATA box (characteristic sequence, rich in A and T)
• core DNA sequence 5'-TATAAA-3' or a variant; usually followed by three or more
adenine bases

• Cis-regulatory elements
– Regions of DNA outside the core promoter that exert control over the promoter
 introns

Polymerase II
complex +1
TATA

Cis-regulatory Element exons

Core Promoter
Structural gene

• The core promoter:

– Sufficient to set the start site and direction of mRNA synthesis
– Basal transcription complex cannot initiate more than low
levels of transcription
– To achieve significant levels of gene expression basal requires
help from transcription factors that bind to Cis-regulatory
elements.
 introns

Polymerase II
complex +1
TATA

Cis-regulatory Element exons

Core Promoter
Structural gene

• Cis- regulatory elements:

– On the same strand of DNA as the gene they control (hence “cis ”)
• May be located close to core promoter (promoter elements) or some distance
away (enhancer elements)

– Interact with the promoter via transcription factors (trans activating

factors) that are generated in response to hormones (or
neurotransmitters, growth factor, cytokines etc.)
– Also referred to as “hormone response elements (HRE)” or “response
elements (RE)”
 Typical transcription factor
Phosphorylation sites

Transcription
activation domain Region involved in protein
dimerization

DNA binding domain

Transcription factors are dimers; each unit has two domains

– DNA binding domain recognises and binds to the specific sequence in RE

– Transcription activation domain; interacts with the basal transcription

complex (or an adaptor protein) to form an active transcription complex

– Contains two sites that can be phosphorylated

– Can be a homodimer or heterodimer

 Transcriptional control
Cis-regulatory element
(Response element)

transcription factor

Polymerase II Structural gene

complex

TATA

promoter

n Transcription factor recognises and binds to RE

n Looping of DNA brings response element close to promoter but in
this state there is no interaction with the basal transcription complex
 Change in
environment
activates signal Transcriptional control
transduction
pathway Cis-regulatory element
(Response element)

kinase kinase
P P
inactive active adaptor transcription
Polymerase II
complex Structural gene
TATA

Mechanism promoter

n Activation of protein kinase

n Protein kinase phosphorylates the transcription factor.
n Phosphorylated transcription factor activates gene expression by interacting
with the basal transcription complex directly or via an adaptor molecule.