Ribosomes

What are the two subunits of ribosomes?

• Small subunit – decodes genetic information from mRNA

• Large subunit catalyses formation of peptide bonds between amino acids to form a polypeptide chain.

What directs the catalytic steps of protein synthesis?

rRNA

What kind of cells have more ribosomes?

Active cells (rapidly growing = high protein synthesis)

What do ribosomes consist of?

rRNA and proteins

What are the roles of the subunits?

Two subunits join on mRNA near 5’ end Ribosome has 4 binding sites for RNA – one for mRNA and three (A,P,E) for tRNA

mRNA pulled through ribosome – 3 nt at a time

tRNAs - adaptors to add each amino acid in the correct sequence

Stop codon – two subunits separate 4 aa per second (bacteria faster)

What is the process of translation?

• Translation - in cytosol on ribosome

• Each amino acid attached to tRNA

• Nucleotides read in sets of three

• Initiated via a start codon recognised by initiator tRNA

• Reaction driven by elongation factors, using GTP hydrolysis

• Process continues until it reaches a stop codon

• Release factor binds to the ribosome – terminates translation and polypeptide is released

• Folding of newly synthesised proteins assisted by chaperones

• Control mechanisms to destroy incorrectly folded proteins

What are the three classes of RNA?

• Messenger RNA (mRNA) - carry the coding sequences for protein synthesis and are called transcripts – carries message from DNA to cytoplasm

• Ribosomal RNA (rRNA) - forms the core of cell's ribosomes

• Transfer RNA (tRNA) - carry amino acids to the ribosomes during protein synthesis

What signals that mRNA ready to leave the nucleus?

Poly-A tail

What is a poly-A tail?

Sequence of adenine nucleotides that is added to the 3' end of the mRNA molecule – polyadenylation

What is RNA polymerase II?

RNA polymerase II synthesises mRNA – requires transcription factors to initiate transcription

What is a mRNA cap?

A highly methylated modification of the 5′ end of RNA pol II-transcribed RNA

• Protects RNA from degradation

• Recruits complexes involved in RNA processing, export and translation initiation

• Marks cellular mRNA as “self” - avoid recognition by innate immune system

What is the transcriptome?

The spectrum of mRNA molecules in a cell.

The transcriptome varies according to cell type and function.

How is transcription regulated?

1. Initiation RNA polymerase and transcription factors bind to the DNA strand at a specific area that facilitates transcription - promoter region Promoter Region often includes specialised nucleotide sequence, TATAAA, (aka - TATA box)

2. Elongation RNA polymerase moves down the DNA template strand in 3' to 5' direction, adding complementary nucleotides Remember – complimentary base pairing

3. Termination and editing Elongation process needs to end and mRNA to separate from DNA template - termination Termination can occur as soon as the polymerase reaches the termination sequence, but in some cases a termination factor (protein) is also needed

What does RNA polymerase III synthesise?

Eukaryotic tRNA

What are tRNA percusors?

tRNAs generally synthesised as larger precursor tRNAs, then modified mature tRNA

• Some tRNA precursors contain introns that must be spliced out - a cut-and-paste mechanism catalysed by endonucleases

• Trimming and splicing require precursor tRNA to be correctly folded in its cloverleaf configuration – quality control process

What happens if RNAs do not pass the control tests?

They are degraded by the nuclear exosome.

How is editing checked?

2 mechanisms –

• Favourable binding - Correct AA has highest affinity for active side pocket of synthetase

• Hydrolytic editing – when tRNA binds, synthetase tries to force adenylated AA into a second editing pocket

How does protein synthesis start?

Initiator tRNA always carries formylmethionine – all newly made proteins have methionine

Initiator tRNA-methionine complex loaded into small ribosomal subunit along with protein – eukaryotic initiation factors (eifs)

Small ribosomal subunit then binds 5’ end of mRNA molecule – moves along mRNA to find AUG Initiation factors dissociate – allowing large ribosomal subunit to assemble with complex and complete ribosome

Initiator tRNA remains at P site, A site vacant and protein synthesis begins

How does protein synthesis stop?

End of coding – one of three stop codons (UAA, UAG, UGA)

Release factors bind to ribosome with stop codon at A site – forces peptidyl transferase to add water molecule instead of aa

Frees carboxyl end of pp chain – chain is released into cytoplasm

Ribosome releases bound mRNA molecule and separates into subunits

New round of synthesis

What are proteins made on?

Polyribosomes

What are the control mechanisms of protein synthesis?

Cell has backup measures to prevent truncated or aberrant protein production.

Broken mRNA – missing either 5’ cap or poly A tail – translation initiation won’t begin Nonsense mediated mRNA decay – prevents mRNA escaping from nuclear envelope.

A nonsense or stop codon in wrong place e.g due to splicing. Happens as mRNA transported to cytosol

What are chaperones?

• Most proteins require molecular chaperones to fold properly

• Many ways to fold unfolded or partially folded proteins – chaperones ensure correct pathway followed

• Many chaperones are heat-shock proteins (hsp) – synthesised in increased amounts when cells exposed to higher temperatures but constitutively expressed.

• Several major families of hsp incl. hsp60 & 70 – function in different organelles e.g. mitochondria contain own hsp60 & hsp70 different from those in cytosol and ER

What is HSP60?

Some proteins cannot fold with Hsp70 alone Hsp60 form barrel shape – an isolation chamber.

Known as a chaperonin

What is a Signal Recognition Particle?

Recognises one class of signal sequence Binds signal sequence on ribosome – transfers entire ribosome and incomplete polypeptide to ER PP + signal sequence moved into ER lumen when synthesised.

In lumen – many glycosylated and moved to Golgi complex, sorted and sent to lysosomes, the plasma membrane, or transport vesicles.

Proteins targeted to the nucleus have an internal signal sequence that is not cleaved once the protein is successfully targeted.

Proteins targeted to mitochondria and chloroplasts in eukaryotic cells use an amino-terminal signal sequence.

Some eukaryotic cells import proteins by receptor-mediated endocytosis.

All cells eventually degrade proteins, using specializes proteolytic systems.

What is the carboxyl terminus?

A cleavage site, where protease action removes the sequence after the protein is imported into the ER.

If a protein has O-linked glyscosykation, where would it be found?

What is the relevance of the O-?

It is in the endoplasmic reticulum.

It is added to the carboxylic acid end of the amino acid through oxygen.

The pH of the lumen of the golgi apparatus changes ad the compartments mature.

How does it change?

What is the function of this?

It becomes more acidic.

Changes in pH changes the charge of the amino acid in the protein, this causes a shape change. This then enables the cargo to disassociate from its receptor in the membrane.

When vesicles leave their donor compartment they are covered in a “coat” protein, what are the different coat proteins and when are they used?

What is their function?

• COP I - golgi apparatus → ER

• COP II - ER → Golgi

• Clathrin - Golgi to plasma membrane or lysosome

Function - bend the membrane to enable bedding to occur from the donor compartment.

Which subunit of the ribosome contains the mRNA binding groove?

small, 40S

In what direction are proteins synthesised?

N to C, Amino to carboxyl