Translation

Converting mRNA to Protein

DNA mRNA Protein

Information in Sequence of
sequence of amino acids
bases

The basics
 The Message on mRNA
• A group of three bases codes for a specific amino acid
– CODON
– eg, UUU for phenylalanine, CCC for proline, GGG for glycine
• Sixty four different codons
– Includes START and STOP codons
• AUG - start
• UAG, UAA, UGA - stop
– Other 60 easily enough for 20 amino acids
– So what happens to the extra codons?
• THE CODON-AMINO ACID PAIRS ARE CALLED THE
GENETIC CODE
– Code is almost universal
• Slight variations in mitochondria
• Biotechnology would be impossible if the code varied between
species!
The universal genetic code
 Adapter Molecule
• Something that can translate
a small sequence of nucleotides
to an amino acid
• Transfer RNA
– 70-90 nucleotides
– Heavily folded and intra-molecular base
pairs
• Lots of loops
• Often drawn as a ‘clover leaf’
• Sequence to recognise bases
on mRNA
– ANTI-CODON 3’ O

=
• Needs to have amino acid attached 5’ O -C-CH-NH2
– At the 3’ end R
– Amino acid attached to 3’OH tRNA
Aminoacyl
– Synthesis is a two step process
Anti-codon
 tRNA
5’ end
T-psi-C arm 3’ end

acceptor stem

variable loop D arm

anti-codon arms

anti-codon
 Activation of Amino Acid

2P
O
= PP
H2N-CH-C-OH
pyrophosphate
R
Amino acid

=
PPP H2N-CH-C-O- P OH
OH
R
A
A
Aminoacyl
AMP
ATP
 Making the aminoacyl-tRNA
3’ O

=
5’ O -C-CH-NH2
5’ OH 3’
R

tRNA
Anti-codon
Aminoacyl-tRNA

O
P
=

OH
H2N-CH-C-O- P OH
R A
A

Aminoacyl AMP
AMP
 Aminoacyl tRNA synthesis
• Need to make sure that the CORRECT amino acid is
attached to a tRNA
– Incorporation of amino acid is purely based on codon:anti-codon
pairing
– So if the wrong amino acid is on the tRNA then the wrong amino
acid will be put into the protein
• Aminoacyl-tRNA synthetases
– Specific enzyme for each tRNA/amino acid pair
– Have proof reading ability
– Recognise the correct tRNA through nucleotides in the side loops
of tRNA (recognition regions)
• Coupling of the amino acid to tRNA involves an input of
energy
 Using the tRNAs
• The aminoacyl tRNAs attach to the mRNA
– With codon:anti-codon pairing
– Then the amino acids react to form peptide bonds
• Occurs in a stepwise manner
– One tRNA at a time rather than all the tRNAs lining up!
– All catalysed by a large assembly of RNA and protein called the
RIBOSOME
• The ribosome moves down the mRNA
– 5’  3’ direction
– Bringing in one tRNA at a time, forming peptide bonds as it goes
– Protein gets longer as the ribosome moves down the mRNA
 The Machinery
• The ribosome
• Large assembly of RNA and protein that binds
the mRNA and brings in the amino acids
– The RNA is called rRNA
• Very large – measured in Svedbergs
• 30S/40S
– 21 proteins and 16S RNA
• 50S/60S
– 34 proteins and 23S & 5S RNA
• Will spontaneously self assemble from constituent parts
– Proteins form the scaffold, RNAs do the work
 Stages of translation

• Initiation ("beginning"): in this stage, the ribosome gets together

with the mRNA and the first tRNA so translation can begin.

• Elongation ("middle"): in this stage, amino acids are brought to

the ribosome by tRNAs and linked together to form a chain.

• Termination ("end"): in the last stage, the finished polypeptide is

released to go and do its job in the cell.
 aa-tRNA coming into A-site
aa-tRNAs sitting next to each
N other on mRNA

aa-tRNA sitting in P-site

N
N

P P A
new aa-tRNA will
come in to A-site
cycle repeats

N
dipeptide now on
tRNA in P-site peptide bond forms
dipeptide on tRNA in A-site
A
N
P

in fact, the ribosome temporarily ribosome moves

holds the empty tRNA in an E- along – kicking
site before discharge out the blank
tRNA P A
Prokariote polyribosome
 Summary
• With an aminoacyl-tRNA at the P-site
• In comes a new aa-tRNA to the A-site
• A peptide bond forms
– Between the NH2 of the amino acid at the A-site and the carbonyl carbon of the
amino acid at the P-site
• The ribosome moves along the mRNA
– Or at least they move relative to each other!
• The tRNA that was in the A-site is now in the P-site
– And this tRNA is carrying the peptide
• And the old, empty tRNA that used to be in the P-site leaves
– There is a specific exit site for this
• And so a new tRNA comes into the vacant A-site
• Process continues until a stop codon is reached
– no tRNA binds here
– Instead, a release factor (RF) binds and hydrolyses the polypeptide from the
tRNA in the P-site
 Experiment
• Nirenberg and Matthaei
– Early 1960s
• Incubation of cell free extracts
– All 20 amino acids
– One radioactively labeled amino acid
– Synthetic polynucleotide
• Made with polynucleotide phosphorylase
• No template
• Random order based on substrate ratio
– Precipitate proteins made
• AAA  lysine, UUU  phe, CCC  pro
To each tube, add all amino acids

Also, one radioactive

amino acid to each tube

asp glu his lys pro thr phe gli

Add synthetic polynucleotide. Incubate.

Precipitate the protein

Assess how much of the radioactive amino acid has been incoroporated
IMPORTANCE OF DNA REPAIR
DNA repair