BY150

Nature and function of the Gene 2:

Gene expression and regulation

Dr Nigel Brissett
Central dogma of molecular biology
DNA – RNA - Protein

eukaryotes vs prokaryotes
 gene expression:

Transcription

mRNA processing control

Translation

eukaryotes vs prokaryotes
 1. Transcription
from DNA to mRNA

DNA-dependent RNA polymerases (or RNA polymerases)

bacterial RNA polymerase

incorporate nucleotides into strand of RNA from a DNA template

 Structure of bacterial RNA
polymerase
consists of six subunits:
two a
one b
one b’
one w
one s

The s subunit is relatively weakly

bound and can dissociate.

a subunit green
b blue
b’ pink
w yellow
 Prokaryotic transcription

5’ 3’

transcription starts at a transcription halts at a

promoter (‘upstream’) terminator (‘downstream’)
Prokaryotic promoters - some
sequences notice anything?
 prokaryotic promoter sequences are
highly conserved

• Promoters have two conserved sequence regions (red):

–35 element and -10 element (“Pribnow box”)

• Polymerase sigma subunit binds specifically to consensus sequences

Transcription by E. coli RNA polymerase

Polymerase initially
binds non-specifically to
DNA and migrates along
molecule until sigma (s)
interacts with -35 and
-10 promotor elements.

Polymerase unwinds
DNA at initiation site and
transcription begins.

ctd.
Sigma subunit dissociates
from the core polymerase
which migrates along DNA
and elongates the growing
RNA chain

Continues to a termination
point
Transcription termination (stem-loop) in
prokaryotes
Termination is signalled by a
G-C rich inverted repeat
followed by 7 A residues.

A stable ‘stem-loop’ mRNA

structure is formed which
terminates transcription and
mRNA dissociates from DNA
template

alternatively,……..
Transcription termination (stem-loop) in
prokaryotes
Termination is signalled by a
G-C rich inverted repeat
followed by 7 A residues.

A stable ‘stem-loop’ mRNA

structure is formed which
terminates transcription and
mRNA dissociates from DNA
template

Alternatively, transcription of some prokaryotic

genes is terminated by a ‘termination protein’ (Rho)
 Eukaryotic RNA polymerases

(long non-coding)

Yeast RNA polymerase II

12-17 subunits, 9 ‘conserved’
b
similar to bacterial polymerases

RNA Pols I and III: similarities to Pol II but different

mechanisms and transcription factors (see below)

bacterial polymerase
Eukaryotic transcription begins with
formation of a RNA polymerase II
preinitiation complex

• multiple transcription factors

• Protein-DNA interactions
RNA polymerase II preinitiation complex
and transcription initiation
Transcription factor
-25
(TFIID) binds to promoter
consensus sequences
BRE, TATAA etc.
TFIID Recognition via TAFs
(transcription-associated
factors) and TBP (TATA-
binding protein)
 RNA polymerase II preinitiation complex
and transcription initiation
Transcription factor (TFIID)
-25 binds to promoter consensus
sequences
BRE, TATAA etc.
Recognition via TAFs
TFIID (transcription-associated
factors) and TBP (TATA-
binding protein)

RNA polymerase and other transcription

RNA polymerase and other transcription factors
factors (B,E,F,H) bind to form a pre-initiation
(B,E,F,H) bind to form a pre-initiation complex.
complex. Mediator
Mediator protein binds protein binds to complex.
to complex.
 RNA polymerase II preinitiation complex
and transcription initiation
Transcription factor (TFIID)
-25 binds to promoter consensus
sequences
BRE, TATAA etc.
Recognition via TAFs
TFIID (transcription-associated
factors) and TBP (TATA-
binding protein)

Phosphorylation of C-terminal domain of

RNA polymerase and other transcription factors polymerase releases Mediator and other general
(B,E,F,H) bind to form a pre-initiation complex. TFs. Polymerase II catalyses RNA synthesis in
Mediator protein binds to complex. association with other elongation/processing factors
From pre-RNA to mature RNA: mRNA
processing
Prokaryotes- no mRNA processing
(simultaneous transcription/ translation in single compartment)

mRNAs are translated by a

series of multiple ribosomes
called “polysomes”

Eukaryotes- mRNA processing before translation

post-transcriptional processing of
eukaryotic mRNA- 3 stages

initial/primary
transcript or ‘pre-
mRNA’
post-transcriptional processing of
eukaryotic mRNA- 3 stages

initial/primary
transcript or ‘pre-
mRNA’

a. 5’ cap
post-transcriptional processing of
eukaryotic mRNA- 3 stages

initial/primary
transcript or ‘pre-
mRNA’

a. 5’ cap

b. 3’ poly A
tail
post-transcriptional processing of
eukaryotic mRNA- 3 stages

initial/primary
transcript or ‘pre-
mRNA’

a. 5’ cap

b. 3’ poly A
tail

c. intron
splicing
Post-transcriptional mRNA processing I:
5’ capping of mRNA

• A 5′ cap is formed by
the addition of a GTP
in reverse orientation*
to the 5′ end of the
mRNA, forming a 5′-to-
5′ linkage

* ‘normal’ nucleotide • The added G is

phophodiester linkages are 5’- 3’ then methylated
(check what this means if at the N-7 position
unsure)
• methyl groups are
added to the riboses
of the first one or two
5’ Me-G cap: stabilises nucleotides in the
mRNA and translation mRNA
initiation role
Post-transcription mRNA processing II
Addition of poly-A tail
1.Polyadenylation signals:
upstream and downstream
(G-U rich) elements,
AAUAAA sequence
Post-transcription mRNA processing II
Addition of poly-A tail
1.Polyadenylation signals:
upstream and downstream
(G-U rich) elements,
AAUAAA sequence

2.Endonuclease
cleaves pre-
mRNA 10 to 30
nucleotides
downstream of
the AAUAAA,
usually at a CA
sequence
 Post-transcription mRNA processing II
Addition of poly-A tail
1.Polyadenylation signals:
upstream and downstream
(G-U rich) elements,
AAUAAA sequence

2.Endonuclease
cleaves pre-
mRNA 10 to 30
nucleotides
downstream of
the AAUAAA,
usually at a CA
sequence

3. Poly-A
polymerase adds
video animation Poly A tail stabilises mRNA + roles in a poly-A tail of
TheCell7e translation regulation about 200 As to
animation 0802 the 3′ end
Post-transcriptional mRNA processing III

Intron removal

small nuclear ribonuclear

particles (snRNPs) made
introns removed by a
up of small nuclear RNAs
spliceosome complex
(snRNAs) + 6-10 protein
molecules (U1, U2, U4,
U5, U6)
U1 snRNP

Process involves catalytic activity of certain RNA

species (RNA enzymes) also called “ribozymes”
Splicing of pre-mRNA – the
principle
1. Cleavage at 5′
splice site (SS)
and joining of 5′
end of intron to
an A within intron
(branch point).
Splicing of pre-mRNA – the
principle
1. Cleavage at 5′
splice site (SS)
and joining of 5′
end of intron to
an A within intron
(branch point).

2. Cleavage at
the 3’ splice site
and simultaneous
ligation of
exons, resulting
in excision of
intron

http://youtu.be/aVgwr0QpYNE
mRNA to protein: translation
Components of translational process

mRNA
Components of translational process

amino acyl tRNA

mRNA
Components of translational process

amino acyl tRNA

ribosome large unit

mRNA

ribosome small unit

 tRNA structure

Figure 7-28
Essential Cell Note: 70-80 nucleotides long, complementary base pairing (loops),
Biology (©
Garland Science modified bases (eg pseudouridine, dihydrouridine)
2010)
 Linkage of amino
acid to tRNA 1. amino acid
eg alanine
coupled with
AMP
catalysed by family of aminoacyl
tRNA synthetases (@40 tRNA
species/cell)
 Linkage of amino
acid to tRNA 1. amino acid
eg alanine
coupled to
AMP

catalysed by family of aminoacyl

tRNA synthetases (@40 tRNA
species/cell)
2. aminoacyl AMP
combines with
tRNA to form
aminoacyl tRNA
Ribosome- a molecular ‘machine’
which interprets the mRNA code

E.coli: @20,000 per cell (25% dry weight)

Dividing mammalian cell: 1x 107/cell
Ribosomal RNA
structure/sequence

E.coli 16S rRNA

• Structural scaffold for

proteins

• Catalytic activity (eg

peptidyl transferase
of large su)
Ribosomes consist of RNA and
proteins

green 50S protein (light green RNA)

blue 30S protein (light blue RNA)
 Ribosome structure

small subunit
dark green

large subunit
light green

Each ribosome has a binding site for mRNA and

three bindings sites for tRNA (E, P, A sites)
Translation- overview

next session: translation stages

gene regulation
 Key texts
• The following are all good texts that cover the same material. Most
give you MUCH more detail:

– Cooper, G.M. & Hausman, R.E. (2019). The cell, a molecular

approach (8th ed.). Blackwells.
http://www.ncbi.nlm.nih.gov/books/NBK9839/ (2nd Ed)

– Lodish et al. (2012). Molecular Cell Biology, 7th Ed, Freeman;

https://www.ncbi.nlm.nih.gov/books/NBK21475/ (4th Ed)

– Alberts et al. (2015). Molecular Biology of the Cell (6th ed). Garland
Science. http://www.ncbi.nlm.nih.gov/books/NBK21054/ (4th Ed)
 Online resources

Other animations and video tutorials

• DNA learning centre – Cold Spring Harbours online DNA
learning resource - http://www.dnalc.org/

• DNA from the beginning – An animated primer of 75

experiments that made modern genetics
http://www.dnaftb.org/

• Bozeman Science basic video tutorial, Transcription and

translation http://youtu.be/h3b9ArupXZg