Transcript Slides - The University of Sydney

Translation
Lecture 13
Converting mRNA to Protein
DNA
Information in
sequence of
bases
mRNA
Protein
Sequence of
amino acids
How do we convert the sequence of bases in mRNA to a sequence of
amino acids in protein?
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!
– We’ll look at it in more detail later
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’ (eg, fig 9-4)
• Sequence to recognise bases on mRNA
• Needs to have amino acid attached
– At the 3’ end
– Amino acid attached to 3’OH
– Synthesis is a two step process
3’
5’
O
=
– ANTI-CODON
O -C-CH-NH2
R
tRNA
Aminoacyl
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
R
pyrophosphate
Amino acid
=
O
PPP
OH
H2N-CH-C-O- P
OH
R
A
A
Aminoacyl
ATP
AMP
Making the aminoacyl-tRNA
5’
OH 3’
5’
O
N
=
3’
O -C-CH-NH2
3’
R
tRNA
Draw thus..
Aminoacyl-tRNA
Anti-codon
=
O
H2N-CH-C-O- P
OH
R
A
Aminoacyl
AMP
P
OH
A
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
• Note the isoleucine/valine example in the textbook
– 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
– 21 proteins and 16S RNA
• 50S
– 34 proteins and 23S & 5S RNA
• Will spontaneously self assemble from constituent parts
– Proteins form the scaffold, RNAs do the work
aa-tRNA coming into A-site
N
aa-tRNA sitting in P-site
aa-tRNAs sitting next to each
other on mRNA
N
N
A
P
P
new aa-tRNA will
come in to A-site
A
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
holds the empty tRNA in an Esite before discharge
ribosome moves
along – kicking out
the blank tRNA
P
A
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
Text Book
• All of p169-170 – Introduction and Outline
– but don’t worry about the eucaryotic stuff just yet
• p171 is more related to the next lecture but you might as well read it
now
– NOTE THE MISTAKE in Fig 9.2 – the CODING STRAND is not the
directly transcribed strand!!
• p172-174 on tRNA and aminoacyl synthetases
• p175 is really interesting about
– which bits of tRNA are involved in recognising codons
– how proof reading occurs
• p179-180 on Ribosomes
– but don’t worry about eucaryotic ribosomes just yet
• p181 – the process of protein synthesis
– starts with initiation which we will do in the next lecture
– So leap straight to p182-183 and Figure 9-13
– Stopping (with Stop codons!) at the top of page 184
Advanced Only
• 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
– Why didn’t GGG work?
To each tube, add all amino acids
Also, one radioactive
amino acid to each tube
asp glu his lys pro thr phe 等等
Add synthetic polynucleotide. Incubate.
Precipitate the protein
Assess how much of the radioactive amino acid has been incoroporated
More Complex Polymers
• Mixture of 5 parts A to 1 part C
• Random length
– AAA, ACA, AAC, CAA, etc
– With high A containing triplets more likely
• Compare actual degree of incorporation
with predicted frequency of triplets
– Expressed relative to AAA/lysine
Results
Amino acid
Observed
Suggested
Expected
Asp
Glu
His
Pro
Thr
Lys
24
24
6
7
26
100
A2C
A2C
AC2
CCC*, AC2
AC2, CA2
A3
20
20
4
5
24
100
* CCC was already known to be Pro from experiments with poly-C
Later Expts
• Methods for making polynucleotides with
repeating sequences
– CACACACACACACA
– Read as mixture of thr and his
– So if AC2 is his, then CAC must be his
• And ACA must be thr
• Binding of aminoacyl-tRNAs to specific
trinucleotides
– Ribosome, radioactive aa-tRNA and trinucleotides