#25 Protein Translation 2 (mRNA-->protein) 10.22.12 Flashcards
What is aka polyptptide polymerase?
ribosome
what steps does ribosome have to formits polymer product( protein?
initiation, elongatino, termiantion
what is the first step in initiation?
choosing the correct reading frame
What are eIFs?
Eukaryotic Initiation Factors
How is Eukaryotic translation ALWAYS initiated?
With the codon AUG and the special INITIATOR tRNA
in addition to the number of proteins called eukaryotic initiaiton factors, …what else binds to teh intiator tRNA coupled to Met?
small subunit of the riboosome!
wat is the only tRNA htat can bind tightly to the small subunit of the ribosome in teh abasence of the large subunit?
initiator tRNA coupled to Met
Next, the small subunit then movs along the small subunit binds to the 5’ end of the mRNA by contacting what???
eIF4e and eIFG
what does eIF4E do? eIF4G
binds to the 5’ cap.
eiFg bridges eIF4E and PAPB (Poly A Tail Binding Protein)
small subunit then moves along hte mRNA until it reaches the first AUG. This translocatio is asstisted by oter initaition factors that act as…..what?
Helicases! to allow rapid scanning along the mRNA
What happens when the AUG is found?
initiation factors dissociate from the small subunit, allowing the large subunit to bind
What happens to initaitior tRNA?
when AUg is found, inititation factors dissocaite and large subunit binds. As a result, the initaitor tRNA is bound to the P site and the A site is open for an incoming ternary cmplex (amino acyl tRNA bound to GTP and eitherr eukaryotic elongation facto 1 (eEF1 ) or EF-Tu (in bacteria)
what percentage of translation beings at AUG?
90% o the time
depending on teh neighboring sequence
Some sequenes cause the intiation of tRNA to skip to the 2nd or third AUG some of the time. In this way, cells make two or more varieties of the same protein (with and wihtout N-terminal signal sequence, so some is exported and some reatined inside the cell)
What is “leaky scanning”
when some sequences cause the intitiation tRNA to skip to teh second or third AUG , so cells can make 2 or more varieties fo the same protein (wiht or without N-terminal signal sequence, so some is exported and some retained inside the cell”
steps to Transition into Elongation (translation)
- Aminoacytl-tRNA binds
- First peptide bond forms (step 2)
- Elongation Begins
What is the diffference between Bacterial Initiation Factors (IFs) and eIFs? (3)
- No 5’ Cap!
- Shine-Dalgarno Sequence (SDS)
- Other initiation factors involved
Is there a 5’ cap or 3’ poly A tail>in Bacterial IFs?
NO!
If no 5’ cap or 3’ poly A tail in bacteria, how is the correct reading frame selected?
part of rRNA bp with a sequence just upstream from teh AUG start site
Each bacterial mRNA has a sequence just a few nucletodies upstream form teh AUG in the 5’ untranslated region (5’ UTR)
What is the Shine Dalgarno Sequence (SDS or ribosome bindin site)
Ribosome binding site
A bacterial mRNA sequence just upstream form teh AUG in teh 5’ untranslated region (5’UTR)
It base pairs with a complementary site (anti-SDS) on the 16S rRNA of the small subunit to dock the intiatior tRNA into the small subunit P-site in frame with ethe AUG codon
What are the initiation factors that aid in the docking of the 16s rRNA of the small subunit into the small subunit P-site in frame with AUG codon? What other function do these IFs have?
IF1, IF2, IF3, which also recruits the large subunit of the ribosome
what is the initiator tRNA charged with most of the time?
formyl methionin (fMET) but soemtimes different tRNAs are used :
GUG (val)
UUG (Leu)
In polysictronic bacterial messages, each gene needs its own ribosome binding site (Shine-Dalgarno sequence) to initiate translation and its own stop codon to end translation. True or False?
TRUE!
After bindin to CAP, how does eIF2 get to AUG (eukaroties?)
helicase like action motors until it finds AUG –> gTP hydorlysis recruits large subutnit –> message
What is the fucntion of SDS in Bacteria?
positions ribosome so that fMet-tRNA is in the P site
What does IF1 do (bacteria)
blocks A-site along position 1 (since you want fmet on P site)
What does IF3 do>
Blocks binding at E site (want Fmet on AUG)
What consists of an incoming ternary complex (that goes to A-site)
aminoacyl-tRNA bound to GTP and either eEF1 (eukaryotic elongation factor 1) or EF-Tu (elongation factor-2 bacteria)
How does Diphtheria toxin causes diptheria?
Inhibits Translation!!
By catalyzing ADP-ribosylation at one site in eEF2–> thereby inactivating this EF and inhibiting translation
What is the mechanism of the toxin RICIN
from caster beans, inhibits translation by irreversibly cleaving a single specific bond of rRNA to hinder the action of elongation factors
What are the three steps of Elongation?
- tRNA loading
- Peptide Bond Formation
- Translocation
Is Elongation different in Euk and Prok?How?
Not really , but elongation factors have different names
Describe tRNA loading
Loading (to A-site) of ternary complex of GTP, aminoactyl-tRNA and either BACETERIAL EF-TU OR EUKARTYOTIC eEF1
Describe Peptide Bond Formation
amino group of the aa in teh A-site attacks the C-terminal end of hte peptide in teh P-site to form a new peptide bond
what ond is being borken during step 2 peptide bond formation?
Ester bond- ver reactive
what enzymeatic activyt catalyzes peptide bond foramtion?
peptidyl transferse in large ribosomal subunit
what is the active site made of?
entirely of rRNAs (23 S for bacteria and 28 S for eukaryotes), so ribosome s a ribozyme
is the ribosome a ribozyme
yes
Describe the conformational changes in teh ribosome during peptide bond formation>
ribosome ratchets the two subunits into a hybrid state
Describe the steps of TRANSLOCATION (step 3)
ribosomes must move the two tRNAs and the message along
what are the three events of translocation
- mRNA advances by exacly one codon (3 nucleotides) with respect to ribosome -> next codoin sits in A site)
- Deacylated tRNA movesf rom P to E site and the peptidyl-tRNA moves form teh A to P site
- THe ribosome cofnormation is reset
What catalyzes Translocation?
GTP bound to either EF-G (bacterial) or eEF2 (eukaryotic EF2)
What gets hydrolyzed during elongation?
both EF-tu (eEF1) an EF-g (eEF2) are both G proteins that each hydrolyze at least 1 GTP to a GDP dduirng each round of elongation
What catalyzes loading of amnoactyl-tRNA into A-site?
EF-Tu (eEF1)
What ahppens if incorrect aminoactyl-tRNA comes in?
preferentially dissocaites and sometimes hydrolzyes GTP
Binding of correct cognate codon-anticodin paring in A site
triggers conformational chanes in teh ribosome which in turen triggers GTP hydorlysis in EF-TU (eEF1) a
the EF-Tu: GDP complex (eEF1:GDP) …
dissociates and the aminoacylt tRNA productivley docks into teh A-site
What is the decoding process?
sampling different charged tRNA moleuls untilt eh codon and anticodona re compelelmtary=> then peptide bond is formed
What catalyzes translocatoin?
eEF2 or EFG bound to GTP
EF-G:GTP (eEF2:GTP) and hydrolysis of GTP
binds to A-site to accelarate the three step process of translocation
EF-tu and EF-G are molecular mimics
kay
what kind of amino acids mimic phosphodiester bond?
negativley charged Glutamate and Aspartate
What factors recognize the codons?
RELEASE FACTORS (RF)
in bacteria, what recognizes UAA and UAG
RF1
What does RF1 in bacteria recognize
UAA and UAG
Whawt does RF2 in baceteria recognize
UAA and UGA
What recognizes all three stop codons in euk?
eRF1
these RFss bind to A site
What happens when RFs bind to A site?
one end of RF uses aa to recognize stop codon, to mimic the decoding that occurs int eh codon-anticodon interaction
recognition triggers peptride bond hydrolysis at the otehr end of the RF located where the CCA would sit
Why is Glutamine in a conserved Gly-Glygln of RFs?
activates a water moleule to hydrolyze the peptidyl -tRNA, thereby rleasing teh compelted protein form teh ribosome
what happens after the translated protein is released form teh ribosome?
G protein RF3 (or in euk eRF3) binds and by hydrolying GTP to GDP enables rlease of :RF3 (erF3, RF1 or RF2 (or eRF1) and the deacyl -tRNA (the one sititn gin E site)
Wht happens when G rptoein RF3 or eRF3 binds ?
hydrolyzing GTP to GDP –> release of RF3 (erF3) , RF1, R2 (eRF1) and teh deacyl-tRNA
what does eIF3 do with termination?
promotes subunit dissociation and release of mRNA and tRNA (tRNA with gorwing polypeptide chain attached to it)
in bacteria, what is RRF and EF-G/GTP do for termaintion?
they bind t A site
then translocation, aided by GTP hydrolysis releases the two subunits, RRF and EF-G
What does IF3 binding to small subunit (in baceteria) lead to
release of tRNA and mRNA
What is a polyribosome?
having many ribosomes operating simulatneously on a sngle mRNA
what do intiation complexes eIF4E and eIF4G require?
oth 5’ cap and poly A tail
This ensures that translation will only begin on COMPELTE mRNAs so effort will not be wasted on damaged one
what is the energy cost for protein synthesis?
minimum is 4 phosphate equivalents per petpide bond 9at least 2 to add amino adcid to tRNA and at least an additional 2 for each elongation cycle)
extra energy is expended in error corretion by the tRNA synthetase and durign ribosome sampling to find correct aminoactyl-tRNA
What is nonsense mediated mRNA decay?
most powerful surveillance system in eukaryotes
mature mRNA is exported to the cytoplasm with its exon junctions intact (EJCs)
what happens when there is correct splicing?
ribosomes begin translation as soon as mature mRNA enters cytoplasm
helicase activity of riosome is able to “plow” through EJCs (exon jucntion complexes)
upon reachin UAA site termination factors are recruited and nomral terminaton take splace
What happens with incorrect splicing
tirgger onsense mediated mRNA decay
incorreclty spliced genes retain intron with expected stop codons
Translation pauses upon reachin this stop codon –> time to recruit Upf proteins, which in turn triggers mRNA decay
what proteinsn are recruited for nonsense-mediated mRNA decay?
Upf proteins
how many huma proteins incorporate Selenium?
25 human proteins, as selenocystein
Wha tare iodothyronine deiodinases?
medically relevant selenocystein proteins, which activate and deactivate thyroid hormones
what happens with deficienes in selenium
result in cardiomyopathy (Keshan disease)
excess intake is also toxic
UGA stop codon can be sued to code for the 21st amino acid
selenocystein given the correct mRNA stem-loop structure and extra translation factors
how is selenocystein made?
specialized tRNA which recognies UGA stop codon is charged with serine by normal seryl-tRNA synthetatse and the serine is subseqently converted enzymatically to selenocystein. A specific NA structure int eh 3’ UTR of mRNA (step and loop structure) signals that selenocystein is to be inserted at an upstream UGA codon
How are viral reverset transcripatese and integrase produced?
by proteolytic processing of a large protein (Gag-Pol fusion protein) consisting of botht eht Gag and Pol amino acid seqeunces
what is Gag protein?
no frameshift (90% of ribosmes)
What is Gag-Pol Fusion protein
with frameshift (10% of ribosmes)
what are viral capsid proteins prouced by?
proteolytic processign of the more abundant GAg prteoin
gag protein terminates at an in-frame stop codon;
how does frameshit occr
frameshift bypasses this stop codon, allowing syntehsis of longer Gag-ol fusion protein
frameshift occurs b/c features in the locatl RNA structure (inclduign the RNA loopswhon) occausioally cas the tRNA leu attahed to C-terminus of gorwing polypeptide chain to slip backward by one nucleotide after leucine has been incorproated itno rowing chain
what are the final seps to make fucntional proteins?
- Folding (sometimes spontaneous; sometimes with chaperones)
- Post-translatinoal modification (i.e. phosphorylation)
- Assembly (into large complexes with otehr proteins, nucleic acids, and/or cofactors)
What are examples of postranslational modifications?
Phosphorylation (dynamic)
Glycosylation
Hydoxylation
Carboxylation (blood coaguation)
Biotinylate enzyme (TAGs)
Farnesylated protein (Greasy tag that allows soluble protein to be anchored on membrane)
What are most commonly phosphoryalted?
serine/theroine/tyrosine
postranslational carboxy is seen in
blood coag
What are 50S binders?
Macrolides and Chloramphenicols
What are 30S binders?
Aminoglycosides and Tetracylines
What ae Aminoglycosides specific effect?
induce misreading by affectedd decoding center–> messes up fidently by inh peptide trans act
binds 30S
Function of Chloarmpehnicol
Binds 50S
inhibits peptidyl transferase activity
Function of Macrolides
binds 50S
blocks progressino of nascent polypeptide chain by binding to exit tunnel (constipatio) indireclty inhibiting formatino of new peptide bonds
fucntion of tetracyline
block binding of aminoactyl tRNA to A site
binds 30S
