Translation and Post Translational Modification

Elongation Factors

- Elongation Factors (EFs) are proteins that promote movement of ribosome along mRNA using GTP - EFs use the energy of GTP to enhance the efficiency and accuracy of translation by providing "pauses" (e.g. GTP hydrolysis) that allow incorrect base pairs to dissociate

How do antibiotics in terms on protein synthesis?

- Many antibiotics work by inhibiting protein synthesis. - Antibiotics exploit the differences between prokaryotes and eukaryotes. - Antibiotics are natural products of bacteria and fungi that give them a selective advantage over other microbes

Step 4 of protein synthesis on RER

- Once the signal sequence has done its job of getting the protein in to the rER, it is chopped offby signal peptidaseand it's degraded. - You end up with the main protein,which is folded.

Structure of typical mRNA

- RNA is always written as 5' on the left and 3' on the right. • - The 5' cap is a modified base which is 7-Methyl Guanosineandit is the entry site for ribosome. - Next to the cap, at the 5' end, is a non-coding regionknown as 5' UTR (untranslated region). - Then you get the coding region. - Followed by the 3' UTR.] - At the 3' end you get a polyA tailwhich protects the 3' end from degradation

Where in mRNA do ribosomes start scanning from ?

- Ribosome scans from the 5' end (7MeG cap) of the mRNA - Translation starts at the first AUG(Met) and continues in frame.

Describe an example of Post- Translational Modification

- Synthesised on membrane bound ribosomes on rER. - It has a N terminus signal sequencewhich binds to SRP and allows it to be transferred across the membrane of the ER. - Post-Translational Modification of Insulin: 1. Signal sequence is removed and degraded 2. Disulphide bondsform between cysteine residues within the polypeptide chain -3 Disulphide Bonds form while the protein folds to form PROINSULIN (single chain polypeptide with 3 disulphide bonds). 3. Before it is packaged into secretory vesicles, it is proteolytically cleavedintwo positions to release the C chain. 4. This leaves you with active, fully-functional insulin which consists of the A and B chains held together by the three disulphide bonds.

Where does protein synthesis occurs?

- There are several intracellular compartments which have membranes around them e.g. Lysosome, Plasma Membrane, Golgi Apparatus. - Protein synthesis does NOT take place INSIDE any of these organelles. It takes place in the CYTOPLASM. - Slight exception~ protein translation does take place in the mitochondrion

Transfer RNA

- They transport of amino acids to the ribosome. - At least 1 tRNA per amino acid. - Amino Acid is attached to the 3' end. The Amino Acid gets stuck onto the correct tRNA using aminoacyl tRNA synthetases. - There is one aminoacyl tRNA synthetase per amino acid.

General Ribosomes

-Bacterial ribosome is less complex than eukaryotic one it has fewer proteins and fewer RNA. -2 Subunits

Step 1 of Termination

-Recognition of stop codon -Release factors (proteins, not tRNAs) bind to empty A site - there is no tRNA for stop codons

Step 2 of Termination

-Release of polypeptide chain -Once the release factor has bound to the stop codon, peptidyl transferase catalyses transfer of the completed protein chain to water and releases it from the ribosome, by hydrolysing the bond between the t-RNA and the peptide - Then everything dissociates

How do proteins get into membrane bound organelles?

-Secretory and Transmembrane Proteins are synthesised in the Rough Endoplasmic Reticulum. - Proteins that are destined to be secretory or transmembrane have a special sequence (first 20-24 amino acids) called a SIGNAL SEQUENCE. - The signal sequence is present at the N-terminus of the polypeptide(first sequence that is synthesised). - The signal sequence is rich inHYDROPHOBIC AMINO ACIDS (they are the type of amino acids that would like to sit within the lipid bilayer).

Step 3 of initiation

-The complex binds mRNA at AUG, which is complementary to UAC, the anticodon on Met-tRNA, forming hydrogen bonds with it. -This initiator Met-tRNA binding sets the frame for the translation eIF-4E and G bind to the cap and are recognised by the pre-initiation complex

Nature of genetic code

-There are 64 combinations and 20 amino acids occurring in the human body so the code is degenerate. -Common amino acids have several codons. -Rare amino acids have few codons. - Methionine = AUG Start codon - STOP = UAA, UAG, UGA

Summary of Translation

1. Initiation: pre-initiation complex consisting of eIF-2 + GTP + 40S + Met-tRNA binds to 7-MeG cap at 5' end (because eIF4E and G have bound to the cap). Preinitiation complexmoves along mRNA until it reaches the first AUG. GTP hydrolyses to GDP which causes conformational change in preinitiation complex allowing 60S subunit to bind to the complex. eIF-2 and GDP dissociate. 2. Elongation: Another tRNA binds to the A site of the ribosome and peptidyl transferase catalyses the formation of the peptide bond between the two amino acids. First tRNA dissociates and the ribosome moves along. Elongation Factors promote the movement of the ribosome by using energy from GTP. 3. Termination: When the A site moves over a stop codon, a Release Factor binds. Peptidyl transferase then catalyses the transfer of the complete polypeptide chain to water and releases it from the ribosome. Everything then dissociates.

Mechanism by which Aminoacyl tRNA Synthetases work

1. Using energy from ATP, the enzyme forms an intermdiate known as adenylated amino acid. The intermediate has the enzyme bound to AMP (derived from ATP which loses a pyrophospahte molecule) and the AMP is bound to the correct amino acid. 2. The adenylated amino acidthen binds an appropriate tRNA. The AMP comes off and the enzyme dissociates. 3. The amino acid is transferred to the 3' hydroxylof the tRNA.

What are the 3 main stages of translation

1. initiation 2. elongation 3. termination

Step 1 of Elongation

A new tRNA carrying the second amino acid to the A (aminoacyl) site

Step 2 of protein synthesis on RER

Binding of SRP to a receptor at the RER surface, translation resumes

RER after polypeptide has entered

If the protein is destined to be a transmembrane proteinthey will have an extra hydrophobic sequenceto hold them in the membrane.

Post Translational Modification

Modifying proteins after they have been made increases the diversity of the proteins. Types of PTM: - Disulphidebond formation - Proteolytic cleavage - Glycosylation (addition of carbohydrate) - Phosphorylation (addition of phosphate) - Prenylation, Acylation (addition of lipid groups) - Hydroxylation

What direction does translation occur?

Protein translation occurs in a 5' to 3'direction and the protein is synthesised in an N to C directionin the cytoplasm and on the rough ER.

Step 1 of protein synthesis on RER

Recognition of signal sequence by a protein-RNA complex "Signal Recognition Particle" (SRP), halting translation

Step 4 of initiation

The 60S subunit binds, and GTP is hydrolysed to GDP. The eIF-2 and GDP dissociate to be reused. The translation complex and initiation is complete

What diseases can arise due to enzyme mutations?

The enzymes can be mutated in many diseases such as cancers, neuropathies, autoimmune disease and metabolic disease

Step 2 Elongation

The peptide bond is formed between the two amino acids by peptidyl transferase on the 60S subunit

step 2 of initiation

The pre-initiation complex is assembled. This is a complex containing Met-tRNA + initiation factors (eIFs) + 40S subunit: Only Met-tRNA can bind to the 40S subunit alone(this is beacuse others arnt as stable).The 40S subunit is involved in tRNA and mRNA recognition.

Step 1 of intiation

The ribosomal subunits dissociate into the 40S and the 60S

Step 3 of Elongation

The second tRNA is translocated to the P (peptidyl) site and the first tRNA is dissociated Elongation factors are proteins that promote the movement of the ribosome along mRNA using GTP

Step 4 of Elongation

There is a new cycle with a new step 1, repeated, until a stop codon is encountered

Why are aminoacyl synthetases important?

They are important in the fidelity of translation-they make sure that the correct amino acid is bound on to the correct tRNA

Polyribosomes

Translation of a piece of mRNA isn't carried out by one ribosome at a time. There are several ribosomes (polyribosomes) on the mRNA at one time -like a string of beads. This means that much more protein can be produced in the same amount of time.

When does translation stop?

Translation stops at the first IN FRAME stop codon

Step 3 of protein synthesis on RER

Translocation of the growing polypeptide into the lumen of RER

Release Factors

When the ribosome moves to a stop codon, such that the vacant A site is over the stop codon, a group of proteins called RELEASE FACTORS bind to the empty A site.

Charged tRNA

tRNA with amino acid attached - free amino acid to aminoacyl tRNA Synthase - aminoacyl tRNA synthase cleaves pyrophosphate from ATP and binds to remaining AMP to amino acid - Amino a acid is adenylated - Adenylated amino acid is then attached to tRNA - Aminoacyl tRNA Synthase and AMP then detach , leaving the charged tRNA