L11 - Translation Flashcards
The codon facts
Three bases encode an amino acid - codons
Code is non-overlapping and degenerate
- Some amino acids are specified by more than one codon (61 codons and 20 amino acids)
Three possible reading frames from one mRNA
Start codon
AUG = Methionine
Stop codons
UAA UAG UGA - signal end of open reading frame
tRNA base pairing
One end base pairs with codon - anticodon loop
Other end carries the amino acid - 3’ end
Intermolecular base pairing within tRNA gives it structure
Nucleotides within tRNA
Primary sequences of nucleotides varies, even within double stranded regions
Some nucleotides in tRNAs are modified to allow different interactions
What is the ratio of tRNA to codon?
Not a 1:1 ratio
Wobble bases (position 3) allow same anticodon to bind to more than one codon
One way wobble is made is by modification
Types of wobble modification
- Deamination of A to create an inosine
- Inosine can pair to U, C or A
Method of coupling amino acid to the tRNA
- Aminoacyl-tRNA synthetases primes amino acid by adding AMP to C-terminus
- Uses the adenylated amino acid to form aminoacyl-tRNA
- Known as charged tRNA - energy from ATP hydrolysis is contained in ester linkage
What are the two adapters required for translation?
Synthetase
tRNA
Synthetase adapter
Pairs correct amino acid to correct tRNA
They are specific to individual tRNAs
Amino acids have to fit into two pockets in the synthetase - before and after AMP addition
Nucleotides in the anticodon and acceptor stem have pockets in the synthetase
tRNA adapter
Pairs correct codon to correct amino acid within ribosome
Pairing requires specific interactions between molecular surfaces
Ribosome subunits
Two subunits
- Large subunit - catalyzes polymerization
- Small subunit - facilitates tRNA/mRNA interaction
Method of ribosome function
- Subunits come together on 5’ end of mRNA
- Process along mRNA at two amino acids per second
- Separate at the stop codon
How are new amino acids added to the ribosome?
New amino acids are added to the C-terminus of the protein by peptidyl transferase
Replace high energy bond with low energy bond
Method of movement through ribosome
- Charged tRNAs enters A-site
- Petidyl transferase catalyses amino acid addition
- Conformational changes move tRNAs to E- and P-sites and move small subunit three nucleotides
- Uncharged tRNAs leave E-site
How many tRNAs are in the ribosome at once?
2
What is the role of elongation factors?
Help translation and improve accuracy
EF-1 role
Once the anticodon is bound, EF-1 causes two delays before petidyl transferase can act
Hydrolyses GTP to GDP
- More rapid if the codon and anticodon are correctly matched
Dissociates from tRNA
These lags allow time for incorrectly bound tRNAs to fall off
- Some correct tRNAs also fall but at a slower rate
Ribosome structure
Large subunit rRNAs form a structure that contains most of the catalytic activity
- Including petidyl transferase
Riboproteins lie on the surface
Which amino acid tRNA assembles the ribosome
AUG – methionine
Method of ribosome assembly
mRNA that has a cap and tail is bound by eIF-4G and eIF-4E to form a loop
- Checkpoint for broken mRNA
- Eukaryote Initiation Factors
Only the methionine tRNA with eIF-2 can bind to small ribosome subunit alone
- Complex binds to cap and associated initiation factors
- Scans along mRNA and settles on the first AUG
- eIF2 is released and ribosome forms
What are stop codons recognised by?
Recognized by release factors
- Look like charged tRNAs and enter the A-site
- Results in dissociation of the ribosome
How far are ribosomes spaced apart on the polysome?
80 nucleotides
How does protein folding occur?
Folds rapidly putting hydrophobic side chains in the middle
- Achieves a lower energy state
Multistep process - important that steps occur in right order
- Incorrect step may reduce the energy state but block further folding
Misfolded proteins
Generally have exposed hydrophobic regions - lead to aggregation
What is it called when proteins initially fold into roughly the correct confirmation?
Molten globule
What is the role of molecular chaperones?
Reverse incorrect steps in protein folding
What are the two types of heat shock protein?
hsp60 – put misfolded proteins into isolation
hsp70 - bind to exposed hydrophobic amino acids on proteins
How do heat shock proteins work?
High temperatures cause properly folded proteins to unfold
- Chaperones function during normal folding as well as when cell has been overheated
HSP60 family
- Hydrophobic entrance binds to protein partially unfolding it
- GroES cap seals protein inside for 15 seconds to allow refolding
Polyubiquitination marks for destruction in the proteasome
1/3 of all newly synthesized proteins are immediately recycled
Protein aggregates can cause?
Disease
They are large and protease resistant
- Can lead to cell death
- Can cause a chain reaction to misfold more proteins
CJD, Huntington’s and Alzheimer’s
Associated with large, extracellular protein aggregates
Amyloid plaques
Made up of cross-beta filaments
CJD prions
Convert normal proteins
Animals eat infected tissue and some prions enter brain and seed new cross-beta filaments
