Download PG1005 Lecture 18 Translation

TR056/PG1005
Lecture 18
Translation
Dr. Neil Docherty
My Teaching Objectives
•  To explain the meaning of the genetic code
• To introduce tRNA structure and describe the mechanism of
amino acid (AA) charging
• To describe the function of the ribosome in translational
initiation, elongation and termination
Genetic Information Directs
Protein Synthesis From Messenger RNA
5’
Gene code (DNA)
3’
3’
5’
RNA polymerase II
Intermediate (mRNA) 5’
TRANSCRIPTION
3’
NUCLEUS
CYTOPLASM
ribosome (+rRNAs)
membrane
rER-Golgi
TRANSLATION
/TARGETTING
organelle
secretory
vesicle
tRNA (+ amino acid)
free ribosomes
cytoplasmic protein
DNA to mRNA
Transcription Summary
• Chromatin remodelling (e.g. acetylation)
• TATA box binding
• Initiation complex assembly/RNA polymerase II recruitment
• 5’ to 3’ synthesis and 5’ G-capping
• Termination and 3’ poly-A tailing
• Intron removal (splicing)
• Nuclear export
RNA to Protein Paradigm
5’
Mature mRNA
3’
Triplet codes (codons)
Complimentary coding tRNA-AAs (anti-codons)
CODON-ANTI-CODON
MATCHING
AA1 - AA2
PEPTIDE BOND
CATALYSIS
Covalent peptide bond
ON RIBOSOMES
The Genetic Code
• Triplet codes (codons) specify particular amino acids
• Due to a complimentary anticodon found on tRNA charged
with specific amino acids
N.B.
• Redundancy
• Start and Stop Codons
Redundancy most frequent at third base (relevant later)
Reading Frames and The Start Codon
5’
Mature mRNA
3’
Mature mRNA
3’
AUG GUA C
Met Val
5’
AUG GUA C
Trp Tyr
In principle, 3 reading frames exist
However, AUG-Met act as a start signal and sets
reading frame
(It is located by the ribosomal machinery at start of
translation, Hence N-terminal AA always methionine)
Transfer RNA (tRNA)
CODON- ANTICODON-AA
mRNA
tRNA
(80nts post-splicing)
Unusual modified
Bases
e.g. pseudouridine
Wobble Base Pairing (tRNA)
• Several mRNA codons can code
for a specific AA
• Change is mainly due to
differences at 3rd “wobble” position
• The third base of the anticodon on
tRNA is frequently a modified
base
• Modified bases have multiple
binding capacities
• Hence redundancy in codons often
a result third base wobble pairing
between codon and anticodon
Charging of tRNA with AA
Two Steps catalysed by AMINOACYL tRNA SYNTHETASES;
1)  ATP dependent adenylation of AA
2)  Ester linkage of AA to 3’ OH of ribose sugar of 3’ tRNA
nucleotide
Aminoacyl tRNA Synthetases
•  Each AA has its own synthetase (Hence, n=20)
• e.g. One synthetase exists for attachment of tryptophan to a
tRNA, the anti-codon of which is complimentary to the codon
sequence for tryptophan specified in the genetic code
Editing Features of Synthetase Enzymes
Ensure Fidelity of tRNA-AA Matching
What mechanisms allow synthetases to accurately match AA-tRNA
pairs (1:40,000 error rate)?
1)  The correct AA has a high affinity for AA binding pocket of
enzyme (too large=excluded)
2) The correct tRNA is associated with the enzyme by fit of
anti-codon sequences into 3 adjacent nucleotide binding
pockets
3) Adenylated AA is shunted to a new site following tRNA binding.
If it fits, it is hydrolysed (=removed)
The Ribosome
• Two subunit complex translational machinery composed of
Proteins 1/3 and rRNA (2/3).
Charged with the following tasks;
-Matching anti-codons and codons (SMALL SUBUNIT)
-Catalysing peptide bond formation (LARGE SUBUNIT)
LARGE
SMALL
Initiation of Translation
Recall that Met is always the first AA of
a nascent polypeptide
• A specific tRNA called the initiator is
charged with Met
• Initiator is bound to the small ribosomal
subunit in association
eukaryotic initiation factor proteins (EIFs)
• Binding of 5’ cap of mRNA proceeds
• 5’ to 3’ searching of mRNA for first AUG
codon
• EIF disassociation, large subunit binding
• TRANSLATION CAN BEGIN
Ribosomal Binding Sites
Four binding sites important
1)  mRNA
2)  tRNA 1=A (aminoacyl tRNA site)
3)  tRNA 2=P (Peptidyl tRNA site)
4)  tRNA 3=E (tRNA exit site)
Elongation
After step 3 a new
aminoacyl tRNA is
free to bind to A site
Obviously 3’ downstream!
Requires;
1) Elongation
Factors
2) GTP hydrolysis
Catalytic Mechanism
Function of large subunit
23S rRNA driven (ribozyme)
1) Proton abstraction from
amino nitrogen of A site AA
2) Reaction of P site AA carboxyl group
with A site amino nitrogen
1)  Adenine protonation of P site tRNA to release
hydroxylated tRNA
2) Addition of one new peptide bonded AA to
nascent protein
Termination
Translation terminates
at STOP CODONS
UAA
UAG
UGA
No corresponding tRNA
Release Factors bind
H2O added to C-terminal end
Frees chain
mRNA release
Ribosomal subunit disassociation
Your Learning From Today
Should focus on being able to;
1)  Appreciate and explain how nucleotide triplets in mature
mRNA specify a code for amino acids
2) Explain the concept and basis of redundancy in the genetic
Code
3) Describe the structure and function of the ribosome.