L10: protein synthesis

What is the basic concept of translation?

• The cell interprets a genetic message and builds an appropriate polypeptide

• The message is contained in the codons of the mRNA and deciphered by the interpreter (codons in the tRNA)

• mRNA is moved through a ribosome, codons are translated into amino acids one by one

• tRNA codons add amino acid cargo to growing polypeptides when anticodon recognises codon

What amino acid does UUU code for?

Phenylalanine; the first amino acid to be recognised

What is a key part of information transfer?

The accurate pairing of bases by hydrogen bonding

How is each tRNA molecule used?

Used repeatedly, picking up cargo, depositing it at ribosome and leaving to pick up another amino acid

What are the two important recognition steps for accurate transcription between?

1. Between tRNA and amino acid

2. Between tRNA anti-codon and mRNA codon

Which part of the tRNA molecule is the amino acid attachment side?

At the 3 prime end

How many tRNA molecules are there?

There are 20 tRNA molecules as there are 20 amino acids

Role of tRNA?

Transferring amino acids to the site of protein synthesis (ribosome)

How big is a tRNA molecule?

Small nucleic acid around 75-80 nucleotides in length

What differs between tRNA molecules?

There are different ones for each of the 20 different amino acids; each has its distinct sequence of 3 bases, the anti- codon

Common features of tRNA molecules?

1. Overall structure

2. Site of attachment of amino acid (cca at 3 prime end)

What is a wobble?

Some anti-codons can pair with more than one codon: it is in the 3rd base position of the anti- codon

What counteracts the lack of proof reading ability of RNA polymerase?

The anticodon can bind to more than one codon

What is the role of aminoacyl-tRNA synthetase

The enzyme which links a tRNA with the correct amino acid

What does the joining reaction of tRNA to its amino acid require?

A source of energy (ATP)

Process of aminoacyl-tRNA synthetase joining amino acid and tRNA

1. The enzyme has a specific shaped area for the amino acid to attach to (this requires ATP)

2. The tRNA molecule will attach to its specific site on the enzyme

3. AMP is used to join the amino acid and tRNA molecule together

4. The activated amino acid leaves the enzyme

How many enzymes are there for each amino acid?

20: each one has an active site that houses a specific combination of tRNA and amino acid

What is the specificity of enzyme action a key factor in?

Accurate information transfer during translation

What is rRNA an important component of?

Ribosomes

The different areas of a ribosome

• Large subunit

• Small subunit

• E site (exit site)

• P site (where peptide grows)

• A site (acceptor site)

Size and weight of ribosomes?

• Large particles around 20nm

• Visible in electron microscope

• Molecular weight= 2.7x10'6 in prokaryotes

How many subunits do ribosomes have?

2: large 50Svedbert and small 30Svedberg

-they only join together when mRNA is present

How much of ribosome is rRNA and proteins?

65% rRNA and the rest proteins

How can we see the structure of ribosomes in atomic detail?

X ray crystallography

What does rRNA do?

Catalyses the formation of peptide bonds- acts as a ribosome

The key binding sites which the ribosome brings the components for translation together at

• An mRNA binding site (30S subunit)

• A P site (50S subunit)- Peptidyl-tRNA site; binds the growing peptide chain

• an A site (50S subunit)- Aminoacyl-tRNA site; binds the incoming aminoacyl-tRNA

• an E site (50S subunit)- exit site which allows the discharged tRNA to leave the ribosome

The three steps of translation

1. Initiation

2. Elongation

3. Termination

What is the only start codon?

AUG for methionine

Describe translation initiation

• The 30 S subunit of the ribosome binds to the correct site on the mRNA (at the start codon).

• The binding site is specified by the upstream mRNA sequence in prokaryotes and the 5’ cap on the mRNA of eukaryotes

• An initiator tRNA carrying the amino acid methionine binds to the start codon (AUG) on the mRNA.

• The 50S ribosome subunit binds to form the initiation complex

• Protein initiation factors and energy (GTP) are required

What happens during translation initiation

The mature mRNA strand binds to the mRNA binding site on the small ribosomal unit and the initiator tRNA brings the first amino acid into the sequence. GTP energy is used to bring in the large ribosomal unit and the first tRNA molecule is in the P site

Describe translation elongation

Amino acids are added one at a time to the peptide chain to grow the amino acid towards the carboxyl end (N-C)

1. Codon recognition; energy input of 2 GTP molecules is required to bring the correct aminoacyl-tRNA into the A site accurately and efficiently

2. Peptide bond formation; the newly arrived amino acid is joined to the growing peptide chain anchored in the A site. This is catalysed by the rRNA (large subunit)

3. Translocation: the ribosome moves the tRNA in the A site to the P site. The discharged tRNA in the P site is moved to the E site to leave the ribosome which requires energy input of GTP

Translation elongation cycle

• The correct tRNA molecule with the amino acid attached (aminoacyl-tRNA) binds to the A site of the ribosome - this requires energy

• The new amino acid is joined to the growing peptide chain (catalysed by the rRNA) while anchored in the A side

• The Erna is moved from the A site to the P site by the ribosome which moves along 3 bases

• The discharged tRNA is moved from the P site to E site to be released from the ribosome which requires energy

What is required for translation elongation

Various elongation factors (proteins)

Describe translation termination

• Elongation continues until a stop codon appears in the A site of the ribosome (UAA, UAG OR UGA)

• A release factor protein binds and the completed peptide chain is freed by the action of release factor from the P site (adds a water instead of amino acid to end the chain)

• The components of the system come apart from each other and can be used for later translation cycles

What do stop codons code for?

A release factor

How long does it take a single ribosome to make an average size polypeptide?

A minute

What do polyribosomes do?

Many copies of a polypeptide can be made simultaneously by them

What is the code for converting the nucleotide language (4 letters) into amino acid language (20 letters)

A triplet code; 3 nucleotides for one amino acid

Why do we use triplet codes?

• Singlet - 4 possibilities

• Doublet - 16 possibilities

• Triplet - 64 possibilities

• - singlet and doublets would not provide enough possibilities

What is the genetic code?

A triplet non-overlapping code which must be read in the correct reading frame

What is the genetic code and how must it be read?

• It is a triplet, non-overcapping code which must be read in the correct reading frame

• It is degenerate (I.e. shows redundancy but not ambiguity)

What may occur when a polypeptide leaves the ribosome?

• Protein folding- the chain has to fold up to its correct 3D structure required for function

• Other compounds like sugars and lipids may be added and some amino acids might be removed

• The protein may have to be moved to its required location

What confirmed that we use triplet codes?

In the 1950s/60s by the systematic analysis of mutants by a phage

How long did a take to assign all 64 codons to amino acids?

5 years (1960- 65)

What are synonyms

Most amino acids are coded for by >1 codon eg. serine and leucine

Where does protein synthesis start?

Always in the cycosoc

What is a signal sequence

Proteins designated for export out of the cell have a signal sequence of about 20 amino acids at the N -terminus

Stages of post translational modification

1. Polypeptide synthesis begins on a free ribosome

2. A signal recognition particle binds a signal peptide, halting process

3. The SRP binds ER membrane translocation complex

4. SRP leaves while the polypeptide grows through the pore

5. Signal cleaving enzyme cuts off signal peptide

6. The final peptide leaves the ribosome and goes to final location

Different types of mutations

1. base - pair substitution; changes one base for another

2. Missence substitution; changes one base/amino acid

3. Nonsense substitution; causes a premature stop codon

4. Frameshift causing immediate nonsense: premature stop codon due to insertion or deletion of 1 base

5. Frameshift causing extensive missence; insertion or deletion changing all following amino acids

6. Insertion or deletion of 3 nucleotides; extra or missing amino ace

What is DISCI

A mutation causing an immature stop codon

What causes sickle cell haemoglobin

1 base pair change causing the amino acid Glu to change to Val and causes anemia as it causes haemoglobin to bind in on itself