L9: Translation Flashcards
1
Q
Overview
A
- Translation is the production of proteins by ribosomes
- these are made of a small subunit and a large subunit and create poly-peptide chains (with a C-terminal at the end and an N-terminal at the beginning) when they translate mRNA
2
Q
Ribosomes
A
- Bacteria have 30s + 50s subunits = 70s ribosome
- Eukaryotes have a 40s + 60s subunits = 80s ribosome
- Both subunits consist of a mix of ribosomal RNA (rRNA) and proteins
- Both eukaryotes and prokaryotes have more rRNA+protiens in their large subunits than their small ones
- The ‘S’ values are a measure of how heavy their cellular components are: this is found by putting them in a sucrose gradient solution
- The components will form bands based on how heavy they are
- The small/large subunit interface has three sites: E, P and A
- The peptide-transferase activity of the ribosome is the function of RNA (which is unusual, bc usually, it’s enzymes that do this)
3
Q
tRNA + rRNA
A
rRNA forms part of the small + large subunits
- The 16rRNA is a component of the small subunit of E. coli ribosomes
- There’s a lot of self-complementarity and mismatched e.g. G + U
- These tend to occur by the stem-loops
tRNA brings aas to the active site on the ribosome
- It recognizes a codon on the mRNA and brings an aa to the strand
- It has an anticodon area that matches the codon on the mRNA
- It has an acceptor arm w/ 4 unpaired nucleotides ending in -CCA, this is where the aa attaches
- Each tRNA is specific to one aa
4
Q
tRNA specificity + tRNA and aa binding
A
tRNA’s specificity comes from modified bases on the arms:
- the variable loop helps distinguish the different tRNAs
- the modified bases help in specificity e.g. on the D arm
- the last A on the end of tRNA (-CCA) binds covalently through its -OH group to the aa’s -COOH group
- aminoacyl-tRNA synthetase binds the aa to the tRNA (called tRNA-aa charging)
- aa is combined with ATP, a pyrophosphate is removed, so aa is bound to AMP –> the AMP disassociates when tRNA arrives and binds to the aa
5
Q
Prokaryotes - Initiation
A
Step 1: Assembly of the small subunit
- the first tRNA is called the initiator tRNA
- the first aa in a protein is Met
- the initiator tRNA is specific to Met and carries it to the ribosome
- (Bacteria use formyl-Met so their intitator is F-Met initiator tRNA)
- the mRNA has a ribosome binding site which is the recognition site for the small subunit
- this process requires 3 initiation factors and arrive in reverse oder (3, 2, 1)
- the small subunit arrives with initiation factor 3 and binds to the ribsome binding site
- IF3 stops the large subunit from binding too early
- Initiator tRNA arrives accompanied by Met and IF2 and a molecule of GTP
- IF2 lines up the ribsome, the tRNA and the anticodon and the anticodon
- GTP gets hydrolised to GDP. This energy is needed to assemble the large subunit
- This fits over the top of the initiator tRNA and its aa
- Once we get here, the process is irreversible
6
Q
Ribosome Binding Site
A
Sits a few bps upstream of the start codon
- it consits of a consensus sequence to tell the ribosome to bind
- the sequence is called Shine-Dalgarno sequence and is complementary to a similar seqeunce in the 16srRNA (which makes up most of the small subunit)
- so this means it can find the right place on the ribosome
7
Q
Eukaryotes - Initiation
A
- The beginnig of the mRNA is labelled by a 5’-CAP
- this is involved in the ribosomes detection of where to start translating
- the ribosome then finds the start codon by recognising the Kozak sequence which surrounds the start codon
- there are many more IFs
- the polyA tail also assists in the small subunit binding
8
Q
Prokayrotes - Elongation
A
- tRNA’s point of entry into the ribsome is the A site
- the aa it is carrying will then bind to the aa that’s already there in the P site
- the Initiator tRNA starts at the P site right away
- so tRNA will enter the A site along with elongation factor 1A (EF1A)
- EF1A brings along another molecule of GTP
- GTP is hydrolysed and produces the energy to make a peptide bond between the two aas
- the ribosime catalyses this bond formation
- EF1A is expelled, and EF1B regenerates it
- translation is quite costly for the cell to do in terms of energy, which is why post-transcriptional control in important to limit the amount of unnecessary mRNA
9
Q
Prokaryote - Translocation
A
- EF2 with GTP arrives at the ribosme
- GTP is hydryolysed and pushes the tRNAs along one position
- the first tRNA is then deacylated in the E site and is ejected from the ribosome
10
Q
Prokaryotic - Termination
A
- this involes the ribosome recognizing a stop codon which doesn’t specify an aa, but simply termianates translation
- this process involves a release factor
- this looks like an aa-tRNA, so it binds in the A site, but as there’s no aa, so H20 is added onto the end of the chain instead which ends the process and the chain is released
- there are two release factors: RF-1 and RF-2 and they recognise different stop codons
- RF-3 unhooks the chain from all the associated proteins and releases it
- Ribosome recycling factor (RRF) seperates the small and the large subunits so they can be used elsewhere
11
Q
Degeneracy
A
- Several differenct codons can specify the same aa
- there are 30-50 different tRNAs = there are more diff tRNAs than there are aas
- this happens due to ‘wobble’
- this is relaxed base-pairing at the 3rd base in the codon-anti-codon pairing
- so the same tRNA-aa conjugate can be recognised by a few different codons
- this is probably due to the bend that exists in the anticodon
12
Q
Quality control mechanisms
A
- Errors in mRNA could lead to faulty proteins which would be a waste of energy and could also be toxic
- mRNA surveillance mechanisms detect when an incorrect mRNA is being made
- There are two mechanisms:
1. Eukaryotes have nonsense mediated decay (NMD) - this is used to detect and ellimintate mRNA molecule that have premature stop condons which could happen with a mutation or an out-of-sequnce codon
2. Prokaryotes have tmRNA
13
Q
tmRNA
A
- Prokaryotes have a problem with non-stop mRNAs: whan an mRNA is broken and id doesn’t have a stop codon
- so the ribosome will stall when it reaches the end of the mRNA and it can’t dissociate bc there’s not stop codon
- tmRNA (a hybrid between a tRNA and an mRNA) is charged with Ala and a piece of mRNA that has a few condons and its own stop condon
- the Ala will be added to the poly-peptide chain, and the ribosme will cary on and will usually produce a lot more Alas
- when it reaches the stop codon, it can release the chain
- the 10 Alas then act as a tag so the chain can be destroyed because it’s incomplete
