Download Chapter 17: From Gene to Protein 1. Overview of Gene Expression 2. Transcription

Chapter 17:
From Gene to Protein
1. Overview of Gene Expression
2. Transcription
3. The Genetic Code
4. Translation
5. Mutations
1. Overview of Gene Expression
Chapter Reading – pp. 334-337
How are Genes related to DNA?
Genes are segments of DNA that code for a
particular protein (or RNA molecule)
• the human genome contains ~3 billion base
pairs (bps) and ~25,000 genes
• most genes encode proteins
• when we talk about “genes” we will focus on those
that express proteins
• the gene products of some genes are RNA
molecules that play a variety of roles in cells
RESULTS
EXPERIMENT
Growth:
Wild-type
cells growing
and dividing
Classes of Neurospora crassa
No growth:
Mutant cells
cannot grow
and divide
• we now know that
genes code for
more than just
enzymes
Can grow with
or without any
supplements
Can grow on
ornithine,
citrulline, or
arginine
Can grow only
on citrulline or
arginine
Require arginine
to grow
Wild type
Class I mutants
(mutation in
gene A)
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Precursor
Precursor
Precursor
Precursor
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Ornithine
Ornithine
Ornithine
Ornithine
Enzyme B
Enzyme B
Enzyme B
Enzyme B
Citrulline
Citrulline
Citrulline
Citrulline
Enzyme C
Enzyme C
Enzyme C
Enzyme C
Arginine
Arginine
Arginine
Arginine
Condition
MM 
citrulline
MM 
arginine
(control)
Summary
of results
Gene
(codes for
enzyme)
Gene B
Gene C
CONCLUSION
Class II mutants Class III mutants
MM 
ornithine
Gene A
Beadle & Tatum, 1941
Srb & Horowitz, 1944
Class I mutants
Minimal
medium
(MM)
(control)
Minimal medium
• these classic
experiments led to
the “one geneone enzyme”
hypothesis
Wild type
Gene Expression
The expression of most genes involves two
distinct processes:
1) Transcription of a gene into RNA
• RNA is a nucleic acid very similar to DNA
(RNA uses “U” instead of “T”)
• this is essentially creating a “photocopy” of the gene
• occurs in the nucleus
2) Translation of the RNA transcript into protein
• accomplished by ribosomes, in the cytoplasm
Prokaryotes vs Eukaryotes
Nuclear
envelope
RNA
DNA
Protein
DNA
TRANSCRIPTION
RNA PROCESSING
Pre-mRNA
mRNA
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Ribosome
TRANSLATION
Polypeptide
Polypeptide
(a) Bacterial cell
(b) Eukaryotic cell
From DNA to RNA to Protein
DNA
template
strand
5
3
A
C
C
A
A
A
C
C
G
A
G
T
T
G G
T
T
T
G
G
C
T
C
A
3
5
DNA
molecule
Gene 1
TRANSCRIPTION
Gene 2
U
mRNA
G G
U
U
U
G
G
C
U
C
5
A
3
Codon
TRANSLATION
Protein
Trp
Phe
Gly
Ser
Gene 3
Amino acid
• gene expression ends at transcription for genes encoding
RNA gene products
Comparison of DNA & RNA
RNA
DNA
• sugar = Ribose
• sugar = Deoxyribose
• single-stranded
• double-stranded
• A, C, G & U (uracil)
• A, C, G & T (thymine)
2. Transcription
Chapter Reading – pp. 340-345
Stages of
Transcription
Promoter
5
3
Start point
RNA polymerase
INITIATION
RNA polymerase binds
to the promoter of a
gene, separates DNA
Transcription unit
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
2 Elongation
Rewound
DNA
ELONGATION
RNA polymerase makes
RNA complementary to
the template strand
5
3
TERMINATION
5
3
RNA polymerase stops
transcribing when end of
gene is reached
3
5
3
5
RNA
transcript
3 Termination
3
5
5
Completed RNA transcript
3
Direction of transcription (“downstream”)
1 A eukaryotic promoter
Promoter
Nontemplate strand
DNA
5
3
3
5
T A T A A AA
A T AT T T T
TATA box
Transcription
factors
Start point
Template strand
2 Several transcription
factors bind to DNA
5
3
3
5
3 Transcription initiation
complex forms
RNA polymerase II
Transcription factors
5
3
Initiation of
Eukaryotic
Transcription
5
3
RNA transcript
Transcription initiation complex
3
5
Eukaryotic promoters
contain a short
sequence called a
TATA box.
With the help of an
array of transcription
factors, RNA
polymerase binds to
the promoter and
starts transcription.
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
A
3
T
C
C A A
5
3 end
C A
U
C
C A
T
A
G
G T
5
5
C
3
T
Direction of transcription
Template
strand of DNA
Newly made
RNA
ELONGATION
Termination of Transcription
RNA polymerase
released
3
5
RNA transcript released
In self-termination, the transcription of DNA
terminator sequences cause the RNA to fold,
loosening the grip of RNA polymerase on the DNA.
In enzyme-dependent termination, a termination
enzyme pushes between RNA polymerase and
the DNA, releasing the polymerase.
Rho termination
protein
C-G rich
stem-loop
Rho protein moves
along RNA
3
• triggered by stem/loop structure in RNA or termination
factors such as the Rho protein (prokaryotes)
Termination of transcription
Primary RNA Transcripts
Newly made RNA molecules in eukaryotes are called
primary transcripts because they need to be
processed before they can carry out their function:
mRNA 1o transcript Protein-coding
segment
Polyadenylation
signal
5
G
P
3
P P
5 Cap
AAUAAA
5 UTR
Start
codon
Stop
codon
3 UTR
AAA … AAA
Poly-A tail
Messenger (mRNA) primary transcripts require the
following modifications:
• a 5’ cap
• a poly-A tail
• splicing out of introns
RNA Splicing
5 Exon Intron
Pre-mRNA 5 Cap
Codon
130
numbers
Exon
Intron
Exon 3
Poly-A tail
31104
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
3 UTR
Coding
segment
The coding region of the primary RNA transcript
contains intervening sequences called introns that
need to be removed or “spliced out”.
The regions that are retained are called exons which
after splicing form a continuous coding region.
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
How is
Splicing
Carried
Out?
Spliceosomes are
structures made of
protein and snRNA
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
snRNA in
spliceosome base
pairs with ends of
intron sequences
resulting in their
being “spliced out”
Exons and Protein Structure
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Exons tend to
code for distinct
substructures
called domains
in proteins.
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Exon shuffling is
thought to be an
evolutionary
process by
which new
genes are made.
Various Roles of RNA Transcripts
1) messenger RNA (mRNA)
• RNA copy of a gene that encodes a polypeptide
2) ribosomal RNA (rRNA)
• RNA that is a structural component of ribosomes
3) transfer RNA (tRNA)
• delivery of “correct”
amino acids to
ribosomes during
translation
For some genes, the end-product
is the RNA itself (rRNA, tRNA)
3. The Genetic Code
Chapter Reading – pp. 337-340
How do Genes code for Proteins?
Recall that proteins are linear polymers
made of 20 different amino acids.
Genes need simply to encode the identity of
each amino acid in a given protein!
• i.e., genes must be capable of encoding
20 different amino acids and their order in a
protein
• although DNA contains only 4 different
nucleotides, this is more than sufficient to
specify 20 different amino acids…
Basis of the Genetic Code
Each amino acid in a protein is specified by
3 nucleotide sequences called codons
• each of the 20 amino acids is coded for by a
unique set of codons:
e.g.
ATG = methionine (start codon)
GGN = glycine
CAA or CAG = glutamine
• there are 64 possible “codon” triplets (4 x 4 x 4)
• more than enough to encode 20 amino acids and the
signal to “stop” or end the protein (TGA, TAA or TAG)
The Genetic Code
If the DNA sequence is:
Second mRNA base
C
A
G
UAU
UGU
U
Cys
Tyr
UAC
UGC
C
U
Ser
UUA
UCA
UAA Stop UGA Stop A
Leu
UUG
UCG
UAG Stop UGG Trp G
UCU
Phe
UUC
UCC
CUU
CCU
U
CAU
CGU
His
CUC
CAC
CGC
C
CCC
C
Leu
Pro
Arg
CUA
CCA
CGA
A
CAA
Gln
CUG
CCG
CGG
G
CAG
AUU
AAU
ACU
AGU
U
Ser
Asn
C
AUC Ile ACC
AAC
AGC
A
Thr
AUA
AAA
ACA
AGA
A
Lys
Arg
or
AUG Met
ACG
AGG
AAG
G
start
GUU
GCU
GAU
U
GUC
GCC
C
GGC
GAC
G
Val
Ala
Gly
GUA
GCA
GGA
A
GAA
Glu
GUG
GCG
GGG
G
GAG
Asp
GGU
5’-CATGCCTGGGCAATAG-3’
3’-GTACGGACCCGTTATC-5’
Third mRNA base (3 end of codon)
First mRNA base (5 end of codon)
U
UUU
(transcription)
The mRNA transcript is:
5’-CAUGCCUGGGCAAUAG-3’
(translation)
The polypeptide is:
*Met-Pro-Gly-Gln (stop)
all proteins begin w/Met
The Genetic Code is Universal
Genes from one species can be expressed in another!
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a jellyfish
gene
4. Translation
Chapter Reading – pp. 83, 345-354
Overview of
Translation
Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
tRNA
C
G
Anticodon
U G G U U U G G C
5
Codons
mRNA
3
Ribosomes facilitate
the production of
polypeptides by:
1) matching codons
in mRNA with
complementary
anticodons in tRNA
2) catalyzing peptide
bonds between
amino acids carried
by tRNAs
Growing
polypeptide
tRNA
molecules
Ribosome
Structure
E P
Exit tunnel
Large
subunit
A
Small
subunit
5
mRNA
3
(a) Computer model of functioning ribosome
Growing polypeptide
P site (Peptidyl-tRNA
binding site)
Exit tunnel
Next amino
acid to be
added to
polypeptide
chain
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
Amino end
P
A
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
E
tRNA
mRNA
5
3
Codons
(c) Schematic model with mRNA and tRNA
Transfer RNA (tRNA)
3
Amino acid
attachment
site
5
tRNA anticodon
will base pair with
a complementary
codon in mRNA
Amino acid
attachment
site
5
3
Hydrogen
bonds
Hydrogen
bonds
A A G
3
Anticodon
Anticodon
(a) Two-dimensional structure
(b) Three-dimensional structure
5
Anticodon
(c) Symbol used
in this book
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
Aminoacyl-tRNA
Synthetases
P Adenosine
P P P Adenosine
P Pi
ATP
Pi
Pi
tRNA
Aminoacyl-tRNA
synthetase
tRNA
Amino
acid
P Adenosine
AMP
Computer model
Aminoacyl tRNA
(“charged tRNA”)
Each tRNA
is loaded
with the
correct
amino acid
due to the
action of
these
enzymes.
Initiation of Translation
Large
ribosomal
subunit
3 U A C 5
5 A U G 3
P site
Pi
Initiator
tRNA

GTP
GDP
E
A
mRNA
5
Start codon
mRNA binding site
3
Small
ribosomal
subunit
5
3
Translation initiation complex
• small ribosomal subunit aligns with start codon of mRNA
• initiator tRNAmet and large subunit then join the complex
Amino end of
polypeptide
E
3
mRNA
Ribosome ready for
next aminoacyl tRNA
P A
site site
5
Elongation
Cycle of
Translation
GTP
GDP  P i
E
E
P A
P A
GDP  P i
GTP
E
P A
Termination of Translation
Release
factor
Free
polypeptide
5
3
3
5
5
Stop codon
(UAG, UAA, or UGA)
2
3
GTP
2 GDP  2 P
i
When a stop codon is reached there is no tRNA
with a complementary anticodon.
Instead a release factor fits in the A site and
catalyzes the dissociation of all components.
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
(a)
End of
mRNA
(3 end)
Referred to as
polyribosomes.
Ribosomes
mRNA
(b)
Multiple
Ribosomes
translate the
same mRNA
0.1 m
Ensuring Proper Protein Structure (pg. 83)
Correctly
folded
protein
Polypeptide
Cap
Hollow
cylinder
Chaperonin
(fully assembled)
The cap comes
off, and the properly
folded
protein is 2 The cap attaches, causing the
Steps of Chaperonin
Action:
cylinder to change shape in
released.
such a way that it creates a
1 An unfolded poly-
peptide enters the
cylinder from one end.
hydrophilic environment for
the folding of the polypeptide.
3 The cap comes
off, and the properly
folded protein is
released.
Chaperonins are large protein structures that assist
newly made polypeptides to ensure they fold properly.
• capture improperly folded proteins inside its cavity, forcing
them to unfold and hopefully refold correctly
Translation in the ER
1 Ribosome
5
4
mRNA
Signal
peptide
3
SRP
2
ER
LUMEN
SRP
receptor
protein
Signal
peptide
removed
ER
membrane
Protein
6
CYTOSOL
Translocation
complex
Proteins destined for the “secretory pathway”
have a signal peptide at the N-terminus that
targets the protein to the ER lumen.
Gene Expression in Prokaryotes
RNA polymerase
Transcription and
translation are
not segregated in
prokaryotes.
DNA
mRNA
Polyribosome
RNA
polymerase
Since prokaryotic
RNA transcripts
don’t need to be
processed,
Polyribosome
Polypeptide
translation can
(amino end)
begin before
transcription is
finished!
Direction of
transcription
0.25 m
DNA
Ribosome
mRNA (5 end)
Summary
of Gene
Expression
DNA
TRANSCRIPTION
3
5
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
AminoacyltRNA synthetase
Intron
NUCLEUS
Amino
acid
AMINO ACID
ACTIVATION
tRNA
CYTOPLASM
mRNA
Growing
polypeptide
3
A
Aminoacyl
(charged)
tRNA
P
E
Ribosomal
subunits
TRANSLATION
E
A
Anticodon
Codon
Ribosome
5. Mutation
Chapter Reading – pp. 355-357
Mutations
A mutation is any change in DNA sequence:
• change of one nucleotide to another
• insertion or deletion of nucleotides or DNA fragments
• inversion or recombination of DNA fragments
What causes mutations?
• errors in DNA replication or DNA repair
• chemical mutagenesis
• high energy electromagnetic radiation
• UV light, X-rays, gamma rays
Sickle-cell Anemia
Wild-type hemoglobin
Sickle-cell hemoglobin
Wild-type hemoglobin DNA
C T T
3
5
G A A
5
3
Mutant hemoglobin DNA
C A T
3
G T A
5
mRNA
5
5
3
mRNA
G A A
Normal hemoglobin
Glu
3
5
G U A
Sickle-cell hemoglobin
Val
• a single nucleotide change causes a single amino
acid change resulting in a malformed protein
3
Types of Mutations
Silent mutations:
• have no effect on amino acid specification
Missense mutations:
• result in the change of a single amino acid
Nonsense mutations:
• convert a codon specifying an amino acid to a stop codon
• results in premature truncation of a protein
Insertion/deletion mutations:
• cause a shift in the reading frame of the gene
• all codons downstream of insertion/deletion will be incorrect
Silent Mutations
Wild type
DNA template strand
3 T
A
C
T
T
C
A
A
A
C
C G
A
T
T 5
5 A
T
G
A
A G
T
T
T
G
G C
T
A
A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(a) Nucleotide-pair substitution: silent
A instead of G
3 T
A
C
T
T
C
A
A
A
C
C A
A
T
T 5
5 A
T
G
A
A G
T
T
T
G
G T
T
A
A 3
U instead of C
5 A
U
Met
G
A
A G
Lys
U
U
Phe
U
G
G U
Gly
U
A A 3
Stop
Missense Mutations
Wild type
DNA template strand
3 T
A
C
T
T
C
A
A
A
C
C G
A
T
T 5
5 A
T
G
A
A G
T
T
T
G
G C
T
A
A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(a) Nucleotide-pair substitution: missense
T instead of C
3 T
5 A
A
C
T
T
C
A
A
A
T
C G
A
T
T 5
T
G
A
A G
T
T
T
A
G C
T
A
A 3
U
A A 3
A instead of G
5 A
U
Met
G
A
A G
Lys
U
U
Phe
U
A
G C
Ser
Stop
Nonsense Mutations
Wild type
DNA template strand
3 T
A
C
T
T
C
A
A
A
C
C G
A
T
T 5
5 A
T
G
A
A G
T
T
T
G
G C
T
A
A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(a) Nucleotide-pair substitution: nonsense
T instead of C
A instead of T
3 T
A
C
A
T
C
A
A
A
C
C G
A
T
T 5
5 A
T
G
T
A G
T
T
T
G G C
T
A
A 3
U
U
U
G G C
U
A
A 3
U instead of A
5 A
U
Met
G
U
A G
Stop
Frameshift – Insertion/Deletion
Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T
T
T G G C
T A A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Amino end
Lys
Phe
Gly
Stop
Carboxyl end
(b) Nucleotide-pair insertion or deletion: frameshift causing
immediate nonsense
Extra A
3 T A C A T T C A A A C G G A T T 5
5 A T G T A A G T T T G G C T A A 3
Extra U
5 A U G U A A G U U U G G C U A A 3
Met
Stop
1 nucleotide-pair insertion
Can cause a premature
stop codon…
Frameshift – Insertion/Deletion
Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T
T
T G G C
T A A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Gly
Lys
Phe
Stop
Amino end
Carboxyl end
(b) Nucleotide-pair insertion or deletion: frameshift causing
extensive missense
A missing
…or
extended
shift in
reading
frame…
3 T A C T T C A A C C G A T T 5
5 A T G A A G T T G G C T A A 3
U missing
5 A U G A A G U U G G C U A A
Met
Lys
1 nucleotide-pair deletion
Leu
Ala
3
Frameshift – Insertion/Deletion
Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T
T
T G G C
T A A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Gly
Lys
Phe
Stop
Carboxyl end
Amino end
(b) Nucleotide-pair insertion or deletion: no frameshift, but one
amino acid missing
T T C missing
…or the
addition/
deletion of
a single
amino acid.
3 T A C A A A C C G A T T 5
5 A T G T T T G G C T A A 3
A A G missing
5 A U G
Met
U U U G G C U A A 3
Phe
3 nucleotide-pair deletion
Gly
Stop
Must be a
multiple
of 3!
Key Terms for Chapter 17
• primary transcript, mRNA, tRNA, rRNA, snRNA
• transcription, promoter, TATA box, RNA polymerase
• 5’ cap, poly-A tail, intron, exon, splicing, spliceosome
• rho protein, stem-loop, codon, anti-codon, translation
• aminoacyl tRNA synthetase, polyribosome, signal
peptide, release factor, chaperonin
• mutation: substitution, deletion, insertion
• silent, missense, nonsense mutations
• reading frame, frameshift
Relevant
Chapter
Questions
1-9, 11