7.3 Flashcards
Ribosomes
Ribosomes are made of protein (for stability) and ribosomal RNA (for catalytic activity)
They consist of a large and small subunit:
- The small subunit contains an mRNA binding site
- The large subunit contains three tRNA binding sites – an aminoacyl (A) site, a peptidyl (P) site and an exit (E) site
Ribosomes can be found either freely floating in the cytosol or bound to the rough ER (in eukaryotes)
Ribosomes differ in size in prokaryotes and eukaryotes (prokaryotes = 70S ; eukaryotes = 80S)
Transfer RNA (tRNA)
tRNA molecules fold into a cloverleaf structure with four key regions:
- The acceptor stem (3’-CCA) carries an amino acid
- The anticodon associates with the mRNA codon (via complementary base pairing)
- The T arm associates with the ribosome (via the E, P and A binding sites)
- The D arm associates with the tRNA activating enzyme (responsible for adding the amino acid to the acceptor stem)
Each tRNA molecule binds with a…
specific amino acid in the cytoplasm in a reaction catalysed by a tRNA-activating enzyme
- Each amino acid is recognised by a specific enzyme (the enzyme may recognise multiple tRNA molecules due to degeneracy)
The binding of an amino acid to the tRNA acceptor stem occurs as a result of a two-step process:
- The enzyme binds ATP to the amino acid to form an amino acid–AMP complex linked by a high energy bond (PP released)
- The amino acid is then coupled to tRNA and the AMP is released – the tRNA molecule is now “charged” and ready for use
The function of the ATP (phosphorylation) is
to create a high energy bond that is transferred to the tRNA molecule
- This stored energy will provide the majority of the energy required for peptide bond formation during translation
Initiation
The first stage of translation involves the assembly of the three components that carry out the process (mRNA, tRNA, ribosome)
- The small ribosomal subunit binds to the 5’-end of the mRNA and moves along it until it reaches the start codon (AUG)
- Next, the appropriate tRNA molecule bind to the codon via its anticodon (according to complementary base pairing)
- Finally, the large ribosomal subunit aligns itself to the tRNA molecule at the P site and forms a complex with the small subunit
Elongation
- A second tRNA molecule pairs with the next codon in the ribosomal A site
- The amino acid in the P site is covalently attached via a peptide bond (condensation reaction) to the amino acid in the A site
- The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain
Translocation
- The ribosome moves along the mRNA strand by one codon position (in a 5’ → 3’ direction)
- The deacylated tRNA moves into the E site and is released, while the tRNA carrying the peptide chain moves to the P site
- Another tRNA molecules attaches to the next codon in the now unoccupied A site and the process is repeated
Termination
The final stage of translation involves the disassembly of the components and the release of a polypeptide chain
- Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon
- These codons do not recruit a tRNA molecule, but instead recruit a release factor that signals for translation to stop
- The polypeptide is released and the ribosome disassembles back into its two independent subunits
In eukaryotes, the ribosomes are separated from the genetic material (DNA and RNA) by the…
nucleus
- After transcription, the mRNA must be transported from the nucleus (via nuclear pores) prior to translation by the ribosome
- This transport requires modification to the RNA construct (e.g. 5’-methyl capping and 3’-polyadenylation)
Prokaryotes lack compartmentalised structures (like the nucleus) and so transcription and translation need not be separated
- Ribosomes may begin translating the mRNA molecule while it is still being transcribed from the DNA template
- This is possible because both transcription and translation occur in a 5’ → 3’ direction
A polysome (or a polyribosome) is a…
group of two or more ribosomes translating an mRNA sequence simultaneously
- The polysomes will appear as beads on a string (each ‘bead’ represents a ribosome ; the ‘string’ is the mRNA strand)
- In prokaryotes, the polysomes may form while the mRNA is still being transcribed from the DNA template
- Ribosomes located at the 3’-end of the polysome cluster will have longer polypeptide chains that those at the 5’-end
All proteins produced by eukaryotic cells are initially synthesised by…
ribosomes found freely circulating within the cytosol
- If the protein is targeted for intracellular use within the cytosol, the ribosome remains free and unattached
- If the protein is targeted for secretion, membrane fixation or use in lysosomes, the ribosome becomes bound to the ER
Protein destination is determined by…
the presence or absence of an initial signal sequence on a nascent polypeptide chain
- The presence of this signal sequence results in the recruitment of a signal recognition particle (SRP), which halts translation
- The SRP-ribosome complex then docks at a receptor located on the ER membrane (forming rough ER)
- Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER
- The synthesised protein will then be transported via a vesicle to the Golgi complex (for secretion) or the lysosome
- Proteins targeted for membrane fixation (e.g. integral proteins) get embedded into the ER membrane
- The signal sequence is cleaved and the SRP recycled once the polypeptide is completely synthesised within the ER
Primary (1º) Structure
- The first level of structural organisation in a protein is the order / sequence of amino acids which comprise the polypeptide chain
- The primary structure is formed by covalent peptide bonds between the amine and carboxyl groups of adjacent amino acids
- Primary structure controls all subsequent levels of protein organisation because it determines the nature of the interactions between R groups of different amino acids
