Lesson 4: Protein synthesis and degradation: ribosomes and proteasomes

Ribosomes

Non-membranous organelles where proteins are synthesised

Ribosome Classification

Free ribosomes in the cytoplasm: bound to cytoskelten, synthesize cytoplasmic proteins

Bound to RER: external layer RER membrane, synthesize protein: membrane proteins or secreted proteins

Polyribosomes (in cluster): Cytoplasmic assemblies made up of several ribosomes (5-20 ribosomes) spaced along a single mRNA molecule.

Ribosome Structure

Consist of 2 Subunits which are separated in cytosol but bind for protein synthesis: Small and big Subunit

In prokaryotic cells and Mitochchondria: 70S

In Eukaryotic cells: 80S (60S + 40S+)

Ribosome composition

rRNA (Ribosonal RNA) 60%:

-transcribed from DNA, named nuclear organizer, in nucleolus

-Eukaryotic ribosomes contain 4 rRNA molecules

-Large subunit: 28S

-Small subunit: 18S

Ribosomal proteins 40%:

-translated from their own mRNAin other already existing ribosomes (In cytoplasm)

-Large subunit: -50 proteins

-Small subunit: -30 proteins

Formation of eukaryotic ribosomes

Nucleolus responsible for: rRNA formation, assembly of ribosomal formation

-Inside Nucleolus, Nucleolar organiser region (NOR) contains genes from different regions of one or more chromosomes, that encode for rRNA:

• Nucleolar organizer DNA is transcribed into rRNA.

Which genes does NOR contain

•genes clustered on the short arms of 5 chromosomes: 13, 14, 15, 21 and 22.

• Once transcribed, these rRNAs and imported ribosomal

proteins will be assembled to form the different

ribosomal subunits in the nucleolus.

Formation steps

Synthesis of rRNA and proteins: separately

• rRNA in in the NOR in nucleolus

• Synthesised in cytoplasmic ribosomes → enter nucleus then nucleolus

Formation of 2 subunits: in the nucleolus

• The proteins that have migrated to the nucleolus, assemble with the rRNA to form the two subunits (the large subunit and the small subunit) separately.

Assembly of both subunits: Exported to the cytoplasm

• The two subunits leave the nucleus to the cytoplasm. They remain separate. Both subunits bind only during protein synthesis.

DNA - Desoxyribonucleic acid

-double helix nucleic acid, which contains the genetic

instructions for synthesising other components of the cell (RNA molecules and proteins).

-It is also responsible for hereditary transmission.

Nucleotides: A,T, C, G

RNA – ribonucleic acid Types

single-stranded nucleic acid, may be:

• mRNA – messenger RNA: contains the genetic Information to synthesize the proteins

• rRNA – ribosomal RNA: structure of ribosomes Ribosomes are large complexes of RNA and protein.

• tRNA – transfer RNA: identifies the appropriate amino acids for protein synthesis and brings them to the ribosome

Nucleotides: A, U, C, G

Proteins

Amino acid polymers (polypeptides) bound by peptide bonds.

Primary structure = amino acid chain

Gene

Specific part of DNA that has specific function

Genetic code

From DNA to proteins: Translation machinery

Ribosome: organelle where translation takes place.

• rRNA: part of the Ribosome that:

- enables recognition with mRNA.

- catalyses the reactions of protein synthesis.

– mRNA: indicates the amino acid sequence in the protein being synthesised

– tRNA: transports amino acids to the ribosome depending on the mRNA sequence. It recognise the codon of that amino acid: it serves as a link between mRNA and amino acids.

Ribosome structure

ribosomes contain four binding sites for RNA molecules:

▪ 1 binding site for the mRNA in the small subunit

▪ 3 binding sites for tRNAs in the large subunit:

– A site (aminoacyl-tRNA): where the tRNA with the corresponding amino acid enters

– P site (peptidyl-tRNA): where the peptide is placed (amino acid chain in synthesis)

– E site (exit): where the RNA that has already transferred its amino acid is placed

1. Step: Activation of amino acids

- Recognition between tRNA and amino acid

- Binding of both molecules to form an aminoacyl-tRNA

2. Step: Initiation

-mRNA binds to small subunit of ribosome

-first aminoacyl-tRNA binds small subunit P-site

-Initial codon is AUG and first acid acid is methionine

-large subunit binds to complex

3. Step: Elongation

-elongation of peptide chain

Steps:

1. aminoacyl-tRNA binds to A site of ribosome

2. New peptide Bon formed between amino acid carried by this tRNA and growing amino acid chain

3. Translocation: ribosome moves a codon on mRNA

A-site: empty, P-site: polypeptidic chain, E-site: tRNA without amino acid

4. Step: Termination

-A site enters stop codon of mRNA which is not recognised by any tRNA

-no amino acid for new peptide bond

-polypeptide chain released from ribosome

-both ribosomal subunits separate

Protein syntheses in Eukaryotes

-Tales Place after mRNA maturation (splicing and addition of cap)

-Initation of protein synthesis more complex

-Elongation and termination similar

Protein degradation

Key step to recycle nitrogen and iron from proteins

-proteins in constant production and removal

-amount of protein maintained by synthesis and degradation

Main mechanisms to regulate protein degradation

1. PROTEASOMIC DEGRADATION (UBIQUITIN-PROTEASOME SYSTEM)

2. LYSOSOMAL DEGRADATION (AUTOPHAGY)

Proteasomic degradation

Takes place due to macromolecules complex called Proteasome Which hydralyzes proteins in cytosol

Complex (26S) formed by:

▪ core particle (20S)

▪ two regulatory particles (19S) in the ends.

Degrade: Damaged or oxidised proteins, short-lived proteins

Mechanism

These proteins are labeled by incoparating small protein ubiquitins, proteins recognise ubiqitin and direct the labeled protein towards Proteasome

-ubiquitin capable of polymerising in form of chains

-binds covalently to protein to be degraded via ATP-consumption pathway involving 3 different enzymes (E1-3)

-Labeled proteins interact with regulatory particles

and are degraded by proteolysis in the core

particle.

Dependent of ATP

Lysosomal degradation (Autophagy)

Takes place in lysosomes

-Lysosomes are membrane-enclosed organelles containing hydrolytic enzymes

-responsible for normal destruction and replacement of long-lived proteins: membrane organiser cellular interior proteins that need to be recycled

-damaged proteins, big macrostructures and whole organelles

-also degrade other types of substances: sugars, nucleotides and lipids

Types of lysosome degradation/autophagy

1. Microautophagy: the lysosome incorporates

soluble cytoplasmic proteins. The lysosome engulfs

small amounts of cytoplasm containing the proteins to

be degraded (never big structures)

2. Chaperone-mediated autophagy: lysosomes directly captures the proteins the be degraded by a receptor, called chaperone

3. the lysosome engulfs a vesicle contains large amount of protein or entire organelle