Ch 17 Gene Expression II: Translation mRNA->Protein

1
Ch 17 Gene Expression II Translation
mRNA-gtProtein
2
LE 17-4
Gene 2
Review
DNA molecule
Gene 1
Gene 3
DNA
5
3
DNA strand (template)
TRANSCRIPTION
RNA
3
5
mRNA
Codon
TRANSLATION
Protein
Protein
Amino acid
3
LE 17-4
Gene 2
Review
DNA molecule
Gene 1
Gene 3
DNA
5
3
DNA strand (template)
TRANSCRIPTION
RNA
3
5
mRNA
Codon
TRANSLATION
Protein
Protein
Amino acid
4
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 gt1 codon)

• Codons
• must be read in the correct reading frame in
  order for the specified polypeptide to be produced

5
LE 17-5
Second mRNA base
Codon Table
Genetic Code
First mRNA base (5 end)
Third mRNA base (3 end)
6
Find an example of redundancy in the genetic code.
Which amino acid does not have redundant codons?
Is there a pattern to redundant codons?
7
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

8
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
  aa-tRNA (codon-anticodon interactions)

• Enzyme in ribosome catalyzes peptide bond between
  amino acids
• -gt polypeptide chain grows

9
LE 17-14a
tRNA structure
3
Amino acid attachment site
5
80 nt long
Three different schematics
Hydrogen bonds
In what ways do they convey the same and
different information?
Anticodon
Two-dimensional structure
Amino acid attachment site
5
3
Hydrogen bonds
3
5
Anticodon
Anticodon
Three-dimensional structure
Symbol used in this book
10
LE 17-13
Amino acids
Polypeptide
tRNA with amino acid attached
Ribosome
tRNA
Anticodon
Codons
5
3
mRNA
11
Accurate translation requires two steps

• 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
12
LE 17-15
Amino acid
Aminoacyl-tRNA synthetase (enzyme)
1.
Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA (an activated amino acid)
13
Ribosomes

• Facilitate specific coupling of anticodons with
  codons
• 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

Draw
14
LE 17-16a
Exit tunnel
Growing polypeptide
tRNA molecules
Large subunit
E
P
A
Small subunit
5
3
mRNA
Computer model of functioning ribosome
15
LE 17-16b
Schematic model showing binding sites on ribosome
P site (Peptidyl-tRNA binding site)
A site (Aminoacyl- tRNA binding site)
E site (Exit site)
E
P
A
Large subunit
mRNA binding site
Small subunit
16
LE 17-16c
Amino end
Growing polypeptide
Next amino acid to be added to polypeptide chain
E
tRNA
mRNA
3
Codons
5
Schematic model with mRNA and tRNA
Ribosome translates 5 to 3 on
mRNA. Polypeptide chain grows amino end first,
carboxyl end last.
17
Building a Polypeptide

• The three stages of translation
• Initiation
• Elongation
• Termination

• All three stages require protein translation
  factors

18
Ribosome Association and Initiation of Translation

• 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.
19
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Memorize Start Codon
First mRNA base (5 end)
Third mRNA base (3 end)
20
LE 17-17
Large ribosomal subunit
P site
Met
Met
Initiator tRNA
GTP
GDP
A
E
mRNA
5
5
3
3
Start codon
Small ribosomal subunit
mRNA binding site
Translation initiation complex
21
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

22
LE 17-18
1. Recognition
Amino end of polypeptide
E
3
mRNA
P site
A site
Ribosome ready for next aminoacyl tRNA
5
2
GTP
2 GDP
E
E
P
A
P
A
GDP
GTP
2. Peptide bond formation
3. Translocation
E
P
A
23
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
24
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Memorize Stop Codons
First mRNA base (5 end)
Third mRNA base (3 end)
25
LE 17-19
3
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.
26
Lets 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?
27
3
5
28
Can a transcript (mRNA) be translated by
multiple ribosomes simultaneously?
29
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

30
LE 17-20
Polyribosome or Polysome
Completed polypeptides
Growing polypeptides
Incoming ribosomal subunits
Polyribosome
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).
31
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
32
Polysomes in Prokaryotes
Where and when are transcripts translated in
prokaryotes?
Coupled transcription and translation
33
LE 17-22
RNA polymerase
DNA
mRNA
Polyribosome
Direction of transcription
0.25 mm
RNA polymerase
DNA
Polyribosome
Polypeptide (amino end)
Ribosome
mRNA (5 end)
34
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
35
LE 17-21
Signal peptide targets polypeptides to ER final
polypeptide destined for secretion or membrane
Ribosomes
mRNA
Signal peptide
ER membrane
Signal- recognition particle (SRP)
Signal peptide removed
Protein
SRP receptor protein
CYTOSOL
Translocation complex
ER LUMEN
Is the molecular weight of a secreted protein
different than the predicted translation product
of its mRNA?
36
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-gtgcga -deletion gcca-gtgca -insertion
gcca-gtgacca
Also, breaks, translocations, inversions as
reviewed previously
37
LE 17-23
What kind of mutation? substitution
Wild-type hemoglobin DNA
Mutant hemoglobin DNA
3
5
5
3
Translate into protein
Normal hemoglobin
Sickle-cell hemoglobin
38
LE 17-5
Second mRNA base
Codon Table
Genetic Code
Memorize Start Codon
First mRNA base (5 end)
Third mRNA base (3 end)
39
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?

40
LE 17-24
Wild type
mRNA
5
3
Protein
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
41
Insertions and Deletions

• Alters reading frame -gtframeshift mutation
• Often more devastating than substitutions

42
LE 17-25
Wild type
mRNA
5
3
Protein
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
43
Source of Mutations

• From spontaneous mutations occur during DNA
  replication, recombination, or repair
• From mutagens are physical or chemical agents
  that can cause mutations

44
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

45
LE 17-26
TRANSCRIPTION
DNA
3
RNA polymerase
RNA transcript
5
RNA PROCESSING
Exon
RNA transcript (pre-mRNA)
Intron
Aminoacyl-tRNA synthetase
NUCLEUS
FORMATION OF INITIATION COMPLEX
Amino acid
AMINO ACID ACTIVATION
CYTOPLASM
tRNA
mRNA
Growing polypeptide
Activated amino acid
3
A
P
Ribosomal subunits
E
5
TRANSLATION
E
A
Anticodon
Codon
Ribosome