Translation Machinery and Mechanism- Lectures 26/27 Flashcards
What is tRNA?
the link between mRNA and protein
What are the zones of tRNA?
DUH arm, anticodon arm (anticodon sequence), V loop, T!C arm, discriminator nucleotide, CCA cap (3’ end), and cognate amino acid
What is the “wobble” position?
the third amino acid in a codon that can be variable for some amino acids and does not require the usual complementarity
_____ attaches an amino acid to its cognate tRNA.
aminoactyl tRNA synthetase
What are the key things involved in translation?
tRNA, ribosomes, eukaryotic translation factors
What composes ribosomes?
some protein, but mostly large (40S) and small (60S) subunits that combine to make a 80S which is all composed of rRNA
What are the three eukaryotic translation factors?
eIFs
eEFs
eRFs
What are the steps of translation?
formation of the pre-initiation complex (43S), initiation, elongation, and termination
What are the three sites on a ribosome?
E (exit) site, P (peptidyl) site, A (aminoacytl) site
What composes the pre-initiation complex (43S)?
eIF2 (bound to GTP)
Met-tRNA
40S complex (eIF1,3,5)
What is the sequence of events for initiation?
eIF4 binds to 5’ cap and recruits 43S complex, which scans down mRNA (using ATP) until the AUG (start codon) is found in the proper context; GTP hydrolysis of eIF2 (via eIF5 on the 43S) results in it dissociating from the complex; all other eIF units leave; 80S ribosome component 60S (guided by eIF5B) binds to UAC and Met-tRNA (at the P site); eIF5B leaves the complex
What are the steps of elongation?
next aa-tRNA is escorted to the A site of the ribosome by eEF1A (bound to GTP); hydrolysis of GTP to GDP by GAP on ribosome –> eEF1A leaves complex; peptidyl transferase reaction binds the Met from the Met-tRNA to teh top of the new aa-tRNA; translocation hydrolizes GTP to shift the ribosome so that the Met-tRNA (now cleaved of its Met) is in the E binding site, the new aa-tRNA (now also bound to Met) is in the P binding site, and the A binding site is open for the next aa-tRNA
What regulates which aa-tRNA is used at the P site?
the codon-anticodon interaction between the sequence of the mRNA and the anticodon arm of the tRNA which allows for the correct timing for the hydrolysis reaction to proceed
_____ are formed when multiple ribosomes are translating a single mRNA
polysomes
What induces termination?
a ribosome arriving at a stop codon (this releases it from the mRNA)
Provide examples of common stress signals.
Ca2+ release from ER, LPS, heat shock, TNF-alpha, virus infection, GF deprivation, AA starvation, heme deficiency
How do cells respond to stress signals?
activation of one of a number of protein kinases that phosphorylate eIF2 (initiation factor required to present initiator tRNA to the ribosomal P site)
What happens to cells in response to stress (rapamycin) or poor growth conditions?
mTOR is inactivated, 4E-BP binds to the eIF4E; overall translation initiation is reduced
What happens to cells in the absence of stress (rapamycin) or in good growing conditions or in cancer cells?
mTOR phosphorylates 4E-BP; the eIF4 complex is released; overall translation initiation is increased
What is SUTR regulation?
regulatory proteins that bind to specific UTRs (can be hairpin loop structures formed by intramolecular base pairing) on the mRNA that interfere with scanning
_____ is a naturally-occurring mechanism that cells use to down-regulate specific gene expression via inhibitor of small translation.
RNAi
What can RNAi be used for in research?
introduction of exogenous siRNA allows for “knock-down” of expression of a target gene
