translation Flashcards
translation =
mRNA-dependent protein synthesis where mRNA is used as a template and the enzyme is the ribosome
translation occurs in the…
cytoplasm of eukaryotes
coupled with transcription in prokaryotes
eukaryotic mRNA generated by transcription -
has a single open reading frame (coding sequence) with untranslated regions on either end (5’ and 3’)
eukaryotic translation starts at the…
start of the open reading frame, however, the ribosome doesn’t bind directly to the start site, it binds to the 5’ cap
the ribosome moves…
from the cap and scans the untranslated region to find the start codon at the beginning of the open reading frame
the ribosome doesn’t make anything whilst it scans - it starts reading from the start codon (methionine AUG)
bacteria mRNA =
polycistronic = has multiple translation start sites
protein synthesis occurs on…
polyribosomes
ribosomes bind in sequence from…
5’ start end to the 3’ end
the ribosome goes the whole length of the mRNA, taking its nascent peptide with it an elongating that peptide chain as it goes along
start codon =
AUG
methionine = always the first amino acid at the N-terminus of the growing peptide chain
in some bacterias, GUG is the start codon = valine amino acid which can be used to insert methionine
the peptide chain grows at the…
carboxy-terminus on the ribosome, as the ribosome moves from 5’ end towards 3’ end
stop codon =
UAG
UGA
UAA
transfer RNA:
takes specific amino acid to the ribosome for protein synthesis
at least one tRNA is dedicated to one specific amino acid, although some amino acids have several cognate tRNAs
tRNA contains ‘odd’ nucleosides:
when ribosome gets added to N1 - you get the normal nucleoside uridine
- in all tRNAs: in some cases, Pseudo uridine forms = abnormal rotated base
…due to pseudoisomerase enzyme rotating the base creating an isomer in which uracil is attached to the ribosome via a carbon-carbon at C5 instead of a nitrogen-carbon glycosidic bond at N1
another form of modification of the uracil base is a reduction - in which Dihydrouracil is formed = not a flat ring
secondary structure of tRNA:
- folds into a common secondary structure comprising ~76 nucleotides
- is single-stranded and folds back on itself to form stem-loops with base pairing in the helical stem
- 5’ end carries a monophosphate
- there are 4 helical stem-loops:
DHU-loop = consists of residues of dihydrouracil
anticodon stem-loop = at nucleotides 34, 35, 36
variable stem-loop
T-lop = consists of pdeudo uridine - at nucleotides 54, 55, 56 - 3’ amino acid attachment site (CCA terminus) = at nucleotides 74, 75, 76
tertiary structure of tRNA:
get from secondary structure to the tertiary structure via a process called co-axial stacking to get a characteristic L shape of the tRNA molecule
- unpaired nucleotides in tRNA secondary structure brought together in folded tertiary structure
- tertiary pairs form between partners in the T-loop, D loop and variable stem-loop
- T-loop and D-loop are close in tertiary structure = interactions between unpaired nucleotides in T-loop and D-loop = co-axial stacking
codon-anticodon interctaion:
when tRNA goes into the ribosome it encounters mRNA and undergoes codon-anticodon recognition:
- 3 bases in mRNA codon pair to 3 bases in tRNA anticodon (position 34, 35, 36)
- Watson Crick pairing at first two positions = mRNA codon position 1 and 2 / tRNA anticodon position 2 (35) and 3 (36)
- alternative pairing at mRNA codon position 3 - tRNA anticodon position 1 (34) = wobble pairing
wobble pairing =
- more relaxed form of base pairing where G-U pairs are likely
- G-U mismatch requires wobble geometry
- tRNAs can recognise multiple cognate codons if they have G, U or I in the appropriate position
aminoacyl-tRNA synthase:
aaRS = enzymes that attach appropriate amino acids onto cognate transfer RNA molecules
there are one of these enzymes per amino acid (20 types)
they are named after the cognate amino acid - using the 3 letter abbreviation (e.g. LysRS)
two step process of aminoacyl-tRNA synthases:
1) activation of amino acid:
- the amino acid binds to ATP and the synthase to form an aminoacyl-adenylate, releasing inorganic pyrophosphate
2) charging of cognate tRNA:
- the amino acid from the aminoacyl-adenylate binds to the appropriate tRNA molecule and the synthase gets recycled
how aminoacyl-tRNA synthases find the correct amino acid:
- the amino acid gets added onto the ribose of the terminal adenosine residue (on the CCA terminus) at the end of the tRNA molecule
- aaRS recognises R group of amino acid = have a specific active site to activate a certain amino acid
if an aminoacyl-tRNA synthase is presented with an incorrect amino acid:
false activation of non-cognate amino acid
the tRNA that is cognate for the correct amino acid picks up (with 100% efficiency) the false activated amino acid = forms a false charged intermediate
the false charged intermediate can either:
- false product dissociates from the synthase (non-cognate product forms)
- hydrolytic editing = false intermediate is recognised and cleaved
Fersht ‘double sieve’ model:
the only way to get efficient activation of cognate amino acids is if there was a two-step process = meaning aaRS enzyme must have two active sites:
- activation site
- editing site
the flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site
the amino acid is added onto the end of the tRNA molecule in the activation site - if the amino acid fits well into the editing site, the amino acid is removed by hydrolysis
if the incorrect amino acid is isosteric (same size) to the cognate amino acid, then this normally isn’t a problem as they will be chemically different due to polarity
elongation factor:
carrier protein which takes aminoacyl-tRNA to a ribosome for binding
e. g. elongation factor Tu (GTP binding protein in bacteria):
- when it binds to the ribosome GTP gets hydrolysed and the protein comes out with GDP bound to it
- Tu binds GDP more tightly so a helper protein (Ts) is required to displace the GDP and then gets displaced itself by GTP
the aa-tRNA, elongation factor and GTP complex:
binds to the A site of the ribosome mRNA complex (via codon-anticodon recognition)
ribosomal proofreading:
if the ternary complex is cognate = aa-tRNA is retained in the A site of the ribosome
if the ternary complex is not cognate = aa-tRNA is ejected from the ribosome
sedimentation coefficient/velocity:
shows the structural behaviour of ribosomes when they undergo centrifugation
eukaryotic ribosomes sediment faster than prokaryotic ribosomes (higher s value)
bacterial ribosomes:
70s with two unequal subunits: larger 50s + smaller 30s
larger 50s subunit: contains the peptidyl transferase centre (active site) = peptide bond forming site of the ribosome
smaller 30s subunit: decoding box (active site) = where codons are recognised by aminoacyl-tRNA = codon-anticodon recognition takes place
eukaryotic ribosomes:
80s with two unequal subunits: larger 60s + smaller 40s
what processes are the same in eukaryotes and prokaryotes?
translational elongation and termination
3 binding sites on a ribosome for tRNA:
E/P/A - only two are occupied at any given time (either E/P or P/A)
polypeptide chain elongation:
1) Binding of aminoacyl-tRNA into the A site and initiator amino acid (or during subsequent rounds of elongation, nascent peptidyl-tRNA) in the P site
2) peptide bond formation:
- initiator amino acid/nascent peptidyl-tRNA transferred
from P site onto aminoacyl-tRNA in A site
- the elonagted peptide is now attached to tRNA in A
site and the deacylated (uncharge) tRNA remain in P
site
- catalysed by ribosome itself
3) translocation reaction:
- mRNA must move through the ribosome so that the
next downstream mRNA codon is presented in A site
for decoding during the next elongation cycle
- frees up the A site and moves the ribosome along the
RNA - so its ready to receive the next incoming
aminoacyl-tRNA
- because codons are triplets of nucleotide, such
movement must span exactly 3 mRNA nucleotides to
maintain the reading frame - as the mRNA moves, so
do the tRNA
- deacetylated tRNA is moved from the ribosomal P site
to the E site and peptidyl-tRNA moves from the A site
into the P site = tRNA moves via a two-step reaction:
- first phase = portion in the larger subunit moves
first spontaneously
- second phase = portion in the smaller subunit
moves requiring GTP
after translocation -
the elongation cycle is complete and the next aminoacyl-tRNA can enter the A site to begin a new cycle
the ribosomal A and E site are…
mutually exclusive
- as aminoacyl-tRNA enters the vacant A site, deacylated tRNA is expelled from the E site
polypeptide chain termination:
- stop codons are not recognised by tRNA = chain elongation stops
- the stop codon is recognised by release factor protein and displaced by it (in eukaryotes = ERF, in prokaryotes = RF1 + RF2)
- release factor causes GTP hydrolysis of the bond between the completed peptide chain and tRNA
- newly synthesised protein is released and ribosome is cleaned out and dissociates into subunits, ready to bind to another mRNA
initiation of bacterial translation:
the ribosomal subunit recognises correct AUG start codon because of a sequence within mRNA which the ribosome binds to = Shine-Dalgarno recognition
initiation of bacterial translation:
- the 30s subunit has initiation factors 1 and 3 blocking the A and E site
- initiator tRNA carries initiator amino acid (methionine) to the ribosome - MetRS attaches to the methionine on the initiator tRNA and a transformylase enzyme adds a formyl group to displace an amino group
- initiation factor 2 and GTP also bind
= forming a ternary complex [fmet-tRNAf-IF2-GTP]
- the ternary complex binds to mRNA to generate the 30s initiation complex with the formylmet-tRNA bound to the P site
- 50s subunit joins and triggers GTP hydrolysis and the initiation factors are released
- the resultant 70s initiation complex consists of 70s ribosome, mRNA and initiator fmet-tRNA
``````````````````````` - the initiator aminoacyl-tRNA recognises mRNA start
codon in the ribosomal P site = recognises the correct AUG start codon because of a sequence within mRNA = Shine-Dalgarno recognition (aligns ribosome with the start codon)
how is eukaryotic different from prokaryotic translational initiation?
eukaryotic translation initiation :
- no Shine-Dalgarno recognition (no RNA-RNA interactions
- the small subunit doesn’t bind directly to the initiation codon, it binds to the cap on the mRNA via protein-protein interactions
- more complex = large numbers of initiation factors (eIFs)
- no tranformylase enzyme = no formyl methionine
eukaryotic mRNA:
- capped at the 5’ end and has a tail at the 3’ end
- there is a single open reading frame (coding sequence) between 5’ and 3’ untranslated regions
which eukaryotic initiation factors bind to the small 40s subunit of the ribosome…
eIF1, eIF1A, eIF3, eIF5
which eukaryotic initiation factor bind to the large 60s subunit of the ribosome…
eIF6
which eukaryotic initiation factor bind to the mRNA 5’ cap…
elF4
which eukaryotic initiation factor causes the release of other initiation factors and cleans out other initiation factors that have already done their job…
eIF5
which eukaryotic initiation factor binds to the initiator species of methional tRNA…
elF2
initiation of eukaryotic translation:
1) initiator aminoacyl-tRNA gets picked up by elF2
2) this complex binds to the small 40s ribosomal subunit (which is already bound to eIF1, 1A, 3, 5) = forms 43s PIC (pre-initiation complex)
3) while the PIC is forming, the elF4 binds to the cap of mRNA
4) the PIC also binds to the cap forming a pre-scanning complex
5) PIC scans mRNA until it reaches the start codon at the begging of the open reading frame (elF1 keeps complex open to allow scanning = scanning complex )
6) while PIC it migrates it drags the cap with it causing the mRNA to loop
7) at the end of scanning (when PIC reaches start codon at the P site) elF1 leaves and elF5 moves into its position triggering GTPase activity of elF2, then elF5 leaves
8) the departure of these initiation factors allows the 43 complex to close = complex becomes more compact forming a 48s initiation complex (translation mode)
9) elF5 clears out remaining initiation fcators and brings in GTP = allowing large 60s ribosmal subunit to join to form 80s initiation complex with only elF4 connected to cap
non-standard decoding: stop codon readthrough -
if a nonsense mutation causes stop codon to appear in the frame of the coding sequence:
- most of the time, the ribosome will still go along until it reaches this stop codon = formin incomplete protein chain
- some mutations are leaky = aminoacyl-tRNA accidentally recognises the stop codon and translation continues through the stop codon (e.g. in the lab when bacteria grows where you didn’t expect)
non-standard decoding: programmed frameshifting in E.coli -
e.g. release factor 2 - gene is pfr (protein factor release B)
- when the gene is sequenced, it was found that there was a stop codon within the reading frame
- the ribosome recognises the leucine codon in the P site next to the stop codon in the A site
- the ribosome shifts by one nucleotide (+1 frameshift) - there is no longer a stop codon within the reading frame so translation continues until it stops at the correct stop codon
non-standard decoding: retroviral frameshifting -
- retroviruses infect mammalian cells = RNA virus cause RNA to not be translated
- the viral ssRNA gets converted into ds DNA by reverse transcription catalysed by reverse transcriptase (made as part of a gag-pol fusion, there is a gag/pol overlap = there is a -1 frameshift in order fro the ribosome to translate)
- this dsDNA gets incorporated into the genome of the mammal
e.g. HIV-1 = caused by a retrovirus programmed -1 frameshift
non-standard decoding: ribosomal gymnastics -
e. g. the sequence of the lacZ gene can be changed by the insertion of a small piece of DNA (with a stop codon and a leucine codon) near the beginning of the lacZ gene
- most of the time, the ribosome will stop translation at the inserted stop codon
- some of the time the normal lacZ protein will be formed - this is due to a +6 hop to avoid the stop codon
