what are the steps of translation elongation?
1) tRNA anticodon:codon interaction
2) peptidyl transferase rxn, aka peptide bond formation
what is "dwell" time? its effect?
allows incorporation of the "correct" a.a. in growing peptide chain by charged tRNA w/ codon
longer dwell time per complementarity of anticodon-codon allows GTP-bound eEF1A to hydrolyze GTP, peptide bodn to form
what is eEF1A? what does it do?
small GTP binding protein
brings next tRNA into ribosomal A site
does not leave A site until peptide bond btwn a.a. is made & GTP is hydrolyzed - this is peptidyl transferase rxn
what helps hydrolysis of eEF1A occur?
an eEF1A GAP on the ribosome makes eEF1A-GTP into eEF1a-GDP
what is basis of specificity in a.a. protein synthesis?
dwell time: amt of time charged tRNA spends in the A site, which depends on codon-anticodon interaction
if the codon-anticodon interaction is correct, will spend more time to allow GTP to be hydrolyzed to GDP
how many high energy bonds are hydrolyzed during polypeptide chain synthesis?
4 high energy bonds are hydrolyzed:
2 in aa-tRNA synthetase rxn, 2 in elongation rxn
what is translocation?
once the 2 a.a. in the A site form peptide bond, the next charged tRNA comes into A site;
must move the tRNA w/ its 2 amino acids into P site
this is translocation
what catalyzes translocation?
has GTP hydrolysis to GDP occur, energy req'd for translocation's produced therein
now next charged tRNA can come into A site
what are stop codons?
what recognizes them?
UAA, UAG, UGA
do not code for any a.a.
when this codon's in A site, eRF recognizes it; hydrolyzes, releases whole polypeptide sitting in P site
what is peptide tunnel?
where growing polypeptide chain emerges during process of elongation
emerges N-terminus first
what's a polysome?
an mRNA that has > 1 ribosome translateing it
useful to isolate v. actively translated mRNAs
how do 3' - 5' mRNA ends' interactions occur? what is result?
ribosome arrives at stop codon, released from mRNA
3' end and 5' end of an mRNA are connected via interaction of poly(A) binding protein and eIFS that bind 5' cap
ribosomes that were released from mRNA can rapidly bind again at 5' end of re-initiate translation
"polysomes" form from this, when multiple ribosmes translate a single mRNA, see v. active tln of a particular mRNA by its partitioning into the "polysome fraction"
are bacterial and human ribosomes the same?
no, although structure/fxn of ribosomes is highly conserved throughout life
differences btwn human and bacterial ribosomes enable use of antibiotics that interfere w/ bacterial tln but not w/ human cellular tln
at what levels can tln be regulated?
2) mRNA-specific regulation
why is tln energetically expensive?
4 high energy bonds are broken to add an amino acid into a protein:
1) a tRNA must be hydrolyzed to AMP (= 2 high energy phosphate bonds)
2) GTP is hydrolyzed 2x durning elongation (= 2 high energy phosphate bonds)
how can translation be globally inhibited?
stress signals - starvation, ER stress
these feed into kinases that phosphorylate eIF2
eIF2-phosphorylated gets locked in GDP-bound state so that eIF2B, the eIF GEF, does not work
dsecribe global inhibition of translation via stress signaling
1) stress signal
2) kinase activation
3) eIF2 phosphorylated in its GDP-bound state; locked there
eIF2 is inactive, translation is inhibited b/c eIF2 is an initiation factor required to present initiator tRNA to ribosomal P site, only active in GTP-bound state
describe global activation of translation initiation
eIF4E, part of the eIF4 complex that binds 5' CAP and is required for ribosomes to recognize mRNA for translation initiation
we have 4EBPs, which bind eIF4E and make its interaction w/ rest of eIF4 complex inefficient
binding of eIF4B slows tln initiation
so if get phosphorylation of 4EBPs by kinase (mTOR), they are now 4EBP-P, cannot bind eIF4E
thus get high tln initiation rate
what is mTOR
kinase that is responsible for global translation initiation activation
phospohrylates 4E-BPs, relases them from eIF4E complex, allows increased translation
in very high activity in cancer cells
what is rapamycin?
a drug that inhibits mTOR activity - breaks tln initiation
cancer drug b/c mTOR is often extremely active in cancer cells
how does specific inhibition of mRNA occur?
5' UTR can form a stem loop structure
this can be bound by regulatory protein which recognizes structure
this prevents Kozak scanning by the ribosome
thus inhibits translation for this specific mRNA
what is ferritin? how is ferritin expression regulated?
ferritin = an iron storage protein
abundant iron: IRE-BP does not bind to 5' UTR of ferritin mRNA, translation proceeds
scarce iron: IRE-BP binds ferritin mRNA 5' UTR, translation is blocked
what happens if regulatory protein binds in 3' UTR?
endonuclease is recruited that cleaves message, exposes a 3' end for rapid degradation by 3' endonucleases
method of mRNA-specific regulation
what is miRNA?
how does mechanism work?
miRNA= short, regulatory RNAs that control gene expression by base-pairing w/ specific mRNAs & controlling their stability & tln
naturally-occurring mechanism that cells use to down-regulate specific gene expression via inhibition of translation
small dbl stranded RNAs, miRNAs, are produced in cell from larger precursors
mature miRNA assembles w/ other proteins in RISC complex; single stranded miRNA guides RISC complex to specific target mRNA; binds; targets that mRNA for nuclease degradation
target mRNA is cleaved or tln is inhibited
what is RNAi?
RNA interference: RNA degradation mechanism of the miRNA
presence of foreign, double-stranded RNA in cell triggers RNAi by attracting Dicer, a nuclease in a protein complex
Dicer cleaves the dbl-stranded RNA into short fragments, siRNAs (small interfering RNAs)
the siRNAs then incorporated into RISCs; RISC discards 1 strand of siRNA duplex, uses remaing single stranded RNA to locate a complelmentary foreign RNA molecule
target RNA molecule then rapidly degraded & RISC searches for more foreign RNA molecules
how can one do "knock down" of expression of a target gene?
introduce siRNA (small interfering RNA)
targeted knock-down gene expression
potential pharma took to treat disease
problems w/ siRNA as a pharmaceutical to treat disease?
problems w/ siRNA delivery, lifetime in vivo, and specificity trouble the field
what is drosha?
what happens after it acts?
an endonuclease that works on double stranded RNA
cleaves primary miRNA transcript; releases pre-miRNA
pre-miRNA is piece of RNA that exits from nucleus, into cytoplasm
in cytoplasm, acted on by a 2nd double stranded RNA endonuclease, Dicer
Dicer cuts in 2 places on pre-miRNA, makes miRNA
this miRNA binds to argonaute proteins, forms RISC
what is RISC?
RNA-induced silencing protein complex
part of the RNAi/miRNA mechanism
miRNA assembles w/ other specialized proteins to form a RISC; RISC patrols the cytoplasm, searches for mRNAs that're complementary to the miRNA it carries
when a target mRNA forms base pairs w/ an miRNA, it's destroyed by a nuclease w/in the RISC or tln is blocked
once RISC works on an mRNA molecule, it's released, seeks out other mRNA molecules
where does miRNA regulation work?
developmental pathways for gene expression