Ch 17 Gene Expression II: Translation mRNA->Protein
LE 17-4
Gene 2
Review
DNA
molecule
Gene 1
Gene 3
DNA
DNA strand
(template)
5
3
TRANSCRIPTION
RNA
mRNA
5
3
Codon
TRANSLATION
Protein
Protein
Amino acid
LE 17-4
Gene 2
Review
DNA
molecule
Gene 1
Gene 3
DNA
DNA strand
(template)
5
3
TRANSCRIPTION
RNA
mRNA
5
3
Codon
TRANSLATION
Protein
Protein
Amino acid
Cracking the Code
• 64 codons
– decoded by the mid-1960s
•Genetic code
–redundant but not ambiguous; no codon specifies more
than one amino acid (but one amino acid may have >1
codon)
•Codons
– must be read in the correct reading frame in order for the
specified polypeptide to be produced
LE 17-5
Second mRNA base
Codon Table
Third mRNA base (3 end)
Genetic Code
Find an example of redundancy in the genetic code.
Which amino acid does
not have redundant
codons?
Is there a pattern to
redundant codons?
Evolution of the Genetic Code
• Genetic code
– nearly universal: shared by the simplest bacteria,
plants, fungi and animals
• Genes can be transcribed and translated
after being transferred from one species to
another
Mechanism of Translation
• Ribosomes
- Bind messenger (mRNA)
- Attract transfer RNA (tRNA) to mRNA
- tRNA covalently linked to specific amino acid (aa-tRNA)
-Complementary basepairs form between mRNA and aatRNA (codon-anticodon interactions)
-Enzyme in ribosome catalyzes peptide bond between
amino acids
- -> polypeptide chain grows
LE 17-14a
tRNA structure
3
Amino acid
attachment site
5
~ 80 nt long
Three different schematics
Hydrogen
bonds
Anticodon
Two-dimensional structure
Amino acid
attachment site
5
In what ways do they
convey the same
and different information?
3
Hydrogen
bonds
3
Anticodon
Three-dimensional structure
5
Anticodon
Symbol used in this book
LE 17-13
Amino
acids
Polypeptide
tRNA with
amino acid
attached
Ribosome
tRNA
Anticodon
Codons
5
mRNA
3
Accurate translation requires two steps
1. a correct match between tRNA and an amino acid
- Catalyzed by aminoacyl-tRNA synthetase
2. a correct match between the tRNA anticodon and an mRNA
codon
LE 17-15
Amino acid
Aminoacyl-tRNA
synthetase (enzyme)
1.
Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA
(an “activated
amino acid”)
Ribosomes
• Facilitate specific coupling of anticodons with codons
Draw
• Ribosomal structure
– Two ribosomal subunits (large and small)
• Made of proteins (ribosomal proteins) and
ribosomal RNA (rRNA)
Form binding sites for mRNA and aa-tRNA
LE 17-16a
tRNA
molecules
Growing
polypeptide
Exit tunnel
Large
subunit
E P
A
Small
subunit
5
3
mRNA
Computer model of functioning ribosome
LE 17-16b
Schematic model showing binding sites on ribosome
P site (Peptidyl-tRNA
binding site)
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
P
A
Large
subunit
Small
subunit
LE 17-16c
Amino end
Growing polypeptide
Next amino acid
to be added to
polypeptide chain
E
tRNA
mRNA
5
3
Codons
Schematic model with mRNA and tRNA
Ribosome translates 5’ to 3’ on mRNA.
Polypeptide chain grows amino end first, carboxyl end last.
Building a Polypeptide
• The three stages of translation:
– Initiation
– Elongation
– Termination
•All three stages require protein translation factors
Ribosome Association and Initiation of Translation
1. Small ribosomal subunit binds
mRNA and special initiator tRNA (met-tRNAi)
(carries the amino acid methionine)
2. Small subunit scans along the mRNA until first start codon
(AUG).
3. Initiation factors bring in large subunit
initiator tRNA occupies the P site.
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Third mRNA base (3 end)
Memorize
Start
Codon
LE 17-17
Large
ribosomal
subunit
P site
Initiator tRNA
GTP
GDP
E
A
mRNA
5
3
5
3
Start codon
mRNA binding site
Small
ribosomal
subunit
Translation initiation complex
Elongation of the Polypeptide Chain
- Amino acids are added one by one to the preceding amino
acid
-Elongation factors facilitate
- codon recognition
- peptide bond formation
- translocation
LE 17-18
1. Recognition
Amino end
of polypeptide
E
3
mRNA
Ribosome ready for
next aminoacyl tRNA
P A
site site
5
2
GTP
2 GDP
E
E
P
A
P
A
GDP
GTP
3. Translocation
2. Peptide bond
formation
E
P
A
Termination of Translation
- Occurs when stop codon in mRNA reaches
A site of ribosome
- A site accepts protein called release factor
-Release factor causes addition of water molecule
instead of amino acid
- Polypeptide released, ribosomal subunits dissociate
and fall off mRNA
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Third mRNA base (3 end)
Memorize
Stop
Codons
LE 17-19
Release
factor
Free
polypeptide
5
3
3
3
5
5
Stop codon
(UAG, UAA, or UGA)
When a ribosome reaches a stop
codon on mRNA, the A site of the
ribosome accepts a protein called
a release factor instead of tRNA.
The release factor hydrolyzes the
bond between the tRNA in the
P site and the last amino acid of the
polypeptide chain. The polypeptide
is thus freed from the ribosome.
The two ribosomal subunits
and the other components
of the assembly dissociate.
Let’s translate a mRNA…
5’ cgaggucaaugcccuauguuuagccc 3’
Bracket each codon
in the open reading
frame (ORF).
Write the amino
acid below each
codon.
What is the
anticodon for
the second
codon in
the ORF?
I’m complicated but once
you get to know me
I’m really pretty nice.
Any questions?
5’
3’
Can a transcript (mRNA) be translated by multiple
ribosomes simultaneously?
Polyribosomes
• -a single mRNA (transcript) is translated by
many ribosomes simultaneously
• mRNA+ bound ribosomes= polyribosomes or
polysome
• Allows fast synthesis of many copies a
polypeptide
LE 17-20
Polyribosome
or
Polysome Incoming
Completed
polypeptides
Growing
polypeptides
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.
Ribosomes
mRNA
0.1 mm
This micrograph shows a large polyribosome in a prokaryotic cell (TEM).
Consider:
When a eukaryotic message is transcribed, processed
and transported to the cytosol, is it immediately translated
into protein?
When would a cell need a polypeptide immediately?
When would a cell want to delay translation? Examples?
What strategy could one use to determine whether a
mRNA was being actively translated?
Hint: consider mass
Subject cell homogenate to differential centrifugation
-Heavy polysomes will pellet
-Light untranslated mRNA in supernatant
Polysomes in Prokaryotes
Where and when are transcripts translated in
prokaryotes?
Coupled transcription and translation
LE 17-22
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase
Direction of
transcription
0.25 mm
DNA
Polyribosome
Polypeptide
(amino end)
Ribosome
mRNA (5 end)
Targeting Polypeptides to Specific Locations
In eukaryotes, what are the two populations of
ribosomes?
Free, soluble in cytosol
synthesize soluble proteins
Bound to rER
- synthesize secreted or membrane bound proteins
- tagged with signal peptide at amino end
LE 17-21
Signal peptide targets polypeptides to ER
final polypeptide destined for secretion or membrane
Ribosomes
mRNA
Signal
peptide
Signalrecognition
particle SRP
(SRP)
receptor
ER
membrane
Signal
peptide
removed
Protein
protein
CYTOSOL
ER LUMEN
Translocation
complex
Is the molecular weight of a secreted protein different
than the predicted translation product of its mRNA?
Effect of mutations on gene expression
What is a mutation?
Any change in the genetic material of a cell or
virus
Types of mutations
Point: a single nucleotide change
-substitution gcca->gcga
-deletion gcca->gca
-insertion gcca->gacca
Also, breaks, translocations, inversions as reviewed previously
LE 17-23
What kind of mutation? substitution
Wild-type hemoglobin DNA
3
Mutant hemoglobin DNA
5
3
mRNA
5
mRNA
Transcribe into mRNA
5
3
Normal hemoglobin
5
3
Sickle-cell hemoglobin
Translate into protein
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Third mRNA base (3 end)
Memorize
Start
Codon
Substitutions
• Missense mutations
– Change codon to encode a different amino acid
• Nonsense mutations
– Change codon to encode a stop codon
nearly always leading to a nonfunctional protein
Missense mutations are more common.
Why?
LE 17-24
Wild type
mRNA
5
Protein
3
Stop
Amino end
Carboxyl end
Base-pair substitution
No effect on amino acid sequence
U instead of C
Substitutions
Neutral
Stop
Missense
A instead of G
Change in amino acid
Stop
Nonsense
U instead of A
Premature termination
Stop
Insertions and Deletions
• Alters reading frame ->frameshift mutation
• Often more devastating than substitutions
LE 17-25
Wild type
mRNA 5
Protein
3
Stop
Carboxyl end
Amino end
Base-pair insertion or deletion
Addition frameshift
Extra U
Stop
Deletion frameshift
Missing
Insertion or deletion of 3 nucleotides
Missing
Stop
Source of Mutations
• From spontaneous mutations: occur during
DNA replication, recombination, or repair
• From mutagens are physical or chemical
agents that can cause mutations
What is a gene? revisiting the
question
• A gene is a region of DNA whose final
product is either a polypeptide or an RNA
molecule
LE 17-26
TRANSCRIPTION
DNA
3
5
RNA
polymerase
RNA
transcript
RNA PROCESSING
Exon
RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
CYTOPLASM
FORMATION OF
INITIATION COMPLEX
Amino
acid
AMINO ACID ACTIVATION
tRNA
mRNA
Growing
polypeptide
Activated
amino acid
3
A
P
E
Ribosomal
subunits
5
TRANSLATION
E
A
Codon
Ribosome
Anticodon