Usanee Anukool (Ph.D.

)
Clinical Microbiology
Faculty of Associated Medical Sciences
Chiang Mai University
2 July 2009
Aims
After class, students will be able to:
 Understand the principle of gene expression

 Explain process of gene expression at the

level of transcription and translation

 Identify the differences of prokaryotic and

eukaryotic transcription and ttranslation

2
Contents
Introduction to expression of genetic
information :
 The central dogma in molecular biology

Gene expression at the transcription level

 Transcription in prokaryotes
 Transcription in eukaryotes

Gene expression at the translation level

 Translation in prokaryotes
 Translation in eukaryotes

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Expression of genetic
information
The phenotype of an organism:
Its genes & environmental factors
Table 1. Genome sizes of different
species
Species Number of genes
Phage MS2 4
Phage T4 200
E. coli 4,000
Human 30,000-40,000

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Expression of genetic
information

How do DNAs transmit

genetic information from
generation to generation?
 DNA DNA
 DNA replication

How do the genetic

information is transferred into
phenotype of an organism?
 DNA  Protein
 Transcription to translation
The process of gene
expression
The Central Dogma

Reverse transcription

DNA RNA
Protein Transcription Translation
Replication

Regulations

6
Transcription &
Translation
Transcription
The synthesis of an RNA transcript
complementary to one strand of DNA in gene

Translation
The conversion of genetic information stored
in the nucleotide sequence in RNA transcript
into amino acid sequence of polypeptides

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Transcription
RNA polymerase
DNA RNA molecules
mRNA
tRNA
rRNA
snRNA

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RNA molecules
messenger RNA: mRNAs
The intermediaries that carry genetic
information from DNA to ribosomes when
protein are synthesized

transfer RNA: tRNA

Small RNA molecule that function as adaptors
between amino acids and codons in mRNA
during translation

9
RNA molecules
ribosomal RNA: rRNA
Structural and catalytic components of
ribosomes
 Ribosomes, the intricate machines that
translate nucleotide sequence of mRNAs into
amino acid sequence of polypeptides

small nuclear RNA: snRNA

Structural components of spliceosomes
 Spliceosomes, the nuclear structure that
excise introns from nuclear genes
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Transcription
The mechanism is similar to that of DNA
replication
Excepted that:
The precursors are ribonucleotide
Only one strand of DNA is used as a ‘template
strand’
No requirement of primer

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Transcription
The RNA chain synthesized is
Complementary to the ‘template strand’
Identical to the ‘nontemplate strand’

‘Sense strand’: an mRNA molecule/ the

coding strand of RNA
‘Antisense strand’: an RNA molecule that is
complementary to the mRNA strand

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Transcription

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 Transcription
In General Process:
The transcription stages
2.Initiation
3.Elongation
4.Termination

Key enzyme:
DNA-dependent RNA Polymerase

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Transcription
The RNA polymerase
Initiate at a specific nucleotide sequence
called ‘promoter’
Catalyze the RNA chain elongation (5’
3’)
DNA template
RNA polymerase
n(RTP) (RMP)n + n(PP)

 RTP = ribonucleotide triphosphate

 RMP = ribonucleotide monophosphate

RNA: U replaces T (U=A, C≡G) 15

Transcription in
Prokaryotes
RNA polymerase
Only one polymerase required in prokaryotes
Mutimeric proteins (MW~480,000 Da)

Holoenzyme: 5 polypeptides (2α,β, β’,σ)

 Two are identical (α)
 Thus contains 4 distinct polypeptides with different
functions
Core enzyme: (2α,β, β’) : involved both
initiation and elongation steps
The sigma (σ) factor: involved in the
initiation only
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Transcription in
Prokaryotes
The sigma (σ ) factor
Recognize and bind RNA polymerase to the
transcription initiation/ promoter site in DNA

in vitro experiment:

 Without sigma factor (2α,β, β’), RNA chain initiates
randomly in DNA sequence

 With sigma factor (2α,β, β’,σ ), RNA chain initiates

specifically at sites used in vivo

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Transcription in
Prokaryotes
 Initiation of RNA chain
Binding of RNA pol. to a promoter regions
in DNA
:Two short conserved regions:
 The -10 sequence: TATAAT box
 The -35 sequence: TTGACA box
 The recognition sequence where the sigma
factor initially recognizes and binds to

Localised unwinding of the dsDNA by RNA

pol.

Formation of phosphodiester bonds

between the first few ribo-nts in nascent RNA
chain 18
•Structure of typical promoter in E.coli

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Transcription in
Prokaryotes
Elongation of RNA chain
Extension of RNA chain
Catalyzed by RNA polymerase core enzyme
after release of σ factor
Occur at the ‘transcription bubble’
Unwinding and rewinding by RNA pol. activities

In E. coli:
 average length of a transcription bubble is 18 nt,
and about 40 ribo-nt are incorporated into the
growing chain per second

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•Elongation of RNA chain in E.coli

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Transcription in
Prokaryotes
Termination of RNA chain
The transcription complex dissociated,
releasing nascent RNA
Occur when RNA pol encounters a special
termination signal
called “transcription terminator”
Two types of transcription terminators in E.
coli
 rho (ρ)−dependent terminators
 Require a presence of rho protein
 rho (ρ)− independent terminators
 The sequence contain GC-rich region followed by 6 AT bp
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•Structure of Rho-independent
transcription terminators

DNA

RNA

Folded RNA

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Transcription in
Eukaryotes
More complex process with three classes of RNA
pol. Sensitivity to
Enzymes location Products
α-amanitin

RNA pol. I nucleolus rRNAs, excluding No

5S-rRNA

RNA pol. II nucleus Nuclear pre-mRNAs complete

RNA pol. III nucleus tRNAs, 5s-rRNA, Intermediate

snRNAS

All three RNA pol. requires the assistance of other

proteins called transcription factors
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Transcription in
Eukaryotes
 The majority of the primary transcripts
undergo three major modifications prior to
their transport to the cytoplasm:

1. Addition of 7-methyl guanosine caps at the

5’end
2. Addition Poly(A) tail at the 3’ end
3. Splicing of intron sequence

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•Posttranscriptional processing of RNA
transcript in eukaryotes

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Transcription in
Eukaryotes
Initiation of RNA chain
Initiation complex
 RNA pol.
 Promoter region in RNA
 Transcription factors (TFs)

RNA pol II’s

promoter region: short highly conserved
modules , “the TATA box binding protein,
TBP”
 TATA box : TATAAAA (centered at -30)
 CAAT box : GGCCAATCT (near -80)

TFII-D, A, B, F, E
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•Initiation of transcription by RNA pol. II

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Transcription in
Eukaryotes
RNA chain elongation
Similar to that in prokaryotes

But using three different enzymes:

 RNA Pol I; II & III and addition of 7-methyl-
guanosine (7-MG) caps at 5’- end
(occur when about 30 nt chain long)

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•Addition of 7-methyl-guanosine caps
at 5’-end

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Transcription in
Eukaryotes
Termination
Chain cleavage: occur at downstream
from a polyadenylation signal
‘AAUAAA’ by endonucleolytic
activity

Polyadenylation is catalysed by
poly(A) Polymerase Poly(A) tail
(~ 200 nt long)

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Polyadenylation

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RNA splicing
Removal of intron sequence
Acuracy: exon-GT..…AG-exon (conserved
dinucleotide sequence)

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RNA Splicing
Three types of intron excision
Intron of tRNA precursor
 Endonuclease & ligase activity

Intron of rRNA precursor

 Automatic cleavage (no enzyme involvement)

Intron of mRNA precursor

 carried out by complex ribonucleoprotein particles
called ‘Spliceosomes’
 Spliceosomes: snRNAs-protein complex

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Splicing of tRNA
precursor

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Splicing of rRNA
precursor

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Splicing of mRNA precursor

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Translation
Protein
15% of wet wt. of cells
Play many roles vital to the lives of all cells
Polypeptides: 20 different amino acids
Amino acids:

H H O Carboxyl group
Amino group
H-N - C - C - OH
R

Side
chain
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•The formation of a peptide bond and four
levels of protein organization

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 The Genetic Code
Codon = a unit or word specify one amino acid
or, actually, one aminoacyl-tRNA
Triplets
Non-overlapping
 except rare case: nt are read in 2 different directions
Comma-free
Degenerate:
 Each amino acid is coded by one or more codons
 20 amino acids: 4 different nucleotides, 61 codons
Ordered, usually differing by a single nucleotide
Contains start and stop codons
 AUG (GUG), UAG/UGA/UAA
Nearly universal
 (with minor exception) same meaning in all
organisms
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•The genetic codes

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•Overview of protein synthesis

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Translation
Occurs on ribosomes located in the
cytoplasm
Involves 3 types of RNAs
mRNA, 3-5 rRNA, 40-60 tRNA molecules
tRNA are activated by an aminoacyl tRNA
synthetases
act as adaptor mediating the incorporation
of proper amino acids to polypeptides
Polyribosomes:
each mRNA is simultaneously translated by
several ribosomes
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Translation components
Ribosomes
rRNA-protein complex macromolecules
Two subunits: Large & Small
rRNA synthesis (RNA pol. I) and
ribosomes assembly occur in the
nucleolus

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•Composition of
prokaryotic and
eukaryotic ribosomes

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Translation components
tRNAs
Amino acid attach to tRNA by high energy
bond between carboxyl group of amino acid
and 3’-hydroxyl termini of tRNA
Aminoacyl-tRNA synthetase activated and
charged tRNAs with amino acids in two steps:

1. aa + ATP aa-AMP + Ppi

2. aa-AMP + tRNA aa-tRNA + AMP

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•The tRNA molecules
Nucleotide sequence and Molecular model of yeast
cloverleaf configuration phenylalanine tRNA based on
x-ray diffraction data

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Translation
 The three stages of translation
1. Polypeptide chain initiation
2. Chain elongation
3. Chain termination

 The initiation of translation

 Similar between prokaryotes and eukaryote
in many aspects but there are some
differences

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Translation
The initiation of translation in E. coli
requires:
30S subunit of the ribosome
A special initiator tRNA
 tRNA Met: the translation initiation codon (AUG/GUG)
f
 carries formyl-Methionine
An mRNA molecule
Three soluble protein initiation factors: IF-1, IF-
2, and IF-3
One molecule of GTP
Initiation sequence: “Shine-Dalgarno
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•The initiation translation in E. coli

Shine-Dalgarno sequence Start codon

mRNA 5’...AAC [AGGAGG] AUAUCC AUG UC…3’
16S rRNA 3’…AUGAGAU [UCCUCC] ACUAGG……..5’
Region of base pairing

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•The initiation translation in E. coli

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Translation
The initiation of translation in
eukaryotes
Similar process excepted that:
More complex with many soluble initiation
factors
tRNAiMet
 the methionine is not formylated, i=initiator)
 enter the P site directly (same as in E. coli)

Initiation complex form at 5’ terminus of mRNA,

not at Shine-Dalgarno sequences (Randomly
scan for start codon)
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Translation
The initiation of translation in
eukaryotes (more)
A cap-binding protein (CBP) binds to the 7-
methyl G cap at 5’ terminus in mRNA then
other Ifs bind to CBP-mRNA complex followed
by small (40S) subunit

When AUG is found, IFs are dissociated from

the complex and large subunit (60S) bind to
methyonyl-tRNA/mRNA/40S complex

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 Translation
 The elongation of translation
: basically the same both in prokaryotes and
eukaryotes (similar elongation factors : EF)
 Three steps of elongation (repeated in cyclic
maner)
• Binding of aminoacyl-tRNA to the A site of the
ribosome (EF-Tu/GTP, EF-Ts)
• Transfer of growing peptide chain from tRNA from
the P site to A site by peptide bond formation
catalysed by peptidyl transferase
• Translocation of ribosome along the mRNA

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•Peptide chain elongation in E. coli

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Translation
 The termination of translation
 Occur when the chain termination codons: UAA,
UAG, or UGA enter A site on the ribosome
 Recognized by soluble protein called release
factors (RFs)
 In E. coli :
 RF-1 (UAA, UAG) and RF-2 (UAA, UGA)
 In eukaryotes:
 Single RF (eRF) recognizes all three termination
codons

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•Polypeptide chain termination in E.
coli

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Further readings
 Snustad, D.P., and Simmons, M.J. 2003. In
Principle of Genetics, 3rd ed., pp279-326. John
Willy & Sons, Inc., USA.
 Textbooks in Molecular Genetics, The Cell &
Molecular Biology or Biochemistry

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http://www.web-books.com/MoBio/Free/Ch4E.htm

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3’ 5’

Enhancer (-1000s) Promoter site (TBP, -30 and -80)

upstream control elements (UCEs)

(-200 bases)

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