Translation

Lecture 13

Converting mRNA to Protein

DNA

Information in sequence of bases

Protein

Sequence of amino acids

mRNA

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– ANTI-CODON

• Needs to have amino acid attached– At the 3’ end– Amino acid attached to 3’OH– Synthesis is a two step process

5’

3’

O

Anti-codon

-C-CH-NH2

=

O

R

AminoacyltRNA

tRNA5’ end

3’ end

anti-codon arms

anti-codon

D arm

acceptor stem

variable loop

T-psi-C arm

Activation of Amino Acid

A

P OHH2N-CH-C-O-

=

O

R

AminoacylAMP

A

PPP OH

ATP

H2N-CH-C-OH

=O

R Amino acid

PP

pyrophosphate

2P

Making the aminoacyl-tRNA

5’3’OH

Anti-codon

A

P OHH2N-CH-C-O-

=

O

R

AminoacylAMP

5’

3’

O -C-CH-NH2

=

O

R

A

P OH

AMP

Aminoacyl-tRNA

tRNADraw thus..

3’N

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

NN

N

N

N

aa-tRNA sitting in P-site

aa-tRNA coming into A-siteaa-tRNAs sitting next to each other on mRNA

peptide bond forms

dipeptide on tRNA in A-site

dipeptide now on tRNA in P-site

new aa-tRNA will come in to A-site

cycle repeats

A

A

P

P P A

AP

in fact, the ribosome temporarily holds the empty tRNA in an E-site before discharge

ribosome moves along – kicking out the blank tRNA

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

asp glu his lys pro thr phe 等等

Also, one radioactive amino acid to each tube

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 24 A2C 20

Glu 24 A2C 20

His 6 AC2 4

Pro 7 CCC*, AC2 5

Thr 26 AC2, CA2 24

Lys 100 A3 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