LEC27: Translation Mechanism II & Regulation Flashcards Preview

MCG > LEC27: Translation Mechanism II & Regulation > Flashcards

Flashcards in LEC27: Translation Mechanism II & Regulation Deck (30):
1

what are the steps of translation elongation?

1) tRNA anticodon:codon interaction 

2) peptidyl transferase rxn, aka peptide bond formation

3) translocation 

2

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 

 

3

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

4

what helps hydrolysis of eEF1A occur?

an eEF1A GAP on the ribosome makes eEF1A-GTP into eEF1a-GDP 

 

 

5

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

6

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

7

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

8

what catalyzes translocation?

an EEF 

has GTP hydrolysis to GDP occur, energy req'd for translocation's produced therein 

now next charged tRNA can come into A site

9

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

10

what is peptide tunnel?

where growing polypeptide chain emerges during process of elongation 

emerges N-terminus first 

11

what's a polysome?

an mRNA that has > 1 ribosome translateing it

useful to isolate v. actively translated mRNAs 

 

12

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"

13

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

14

at what levels can tln be regulated?

1) globally 

2) mRNA-specific regulation

15

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)

16

how can translation be globally inhibited?

describe mechanism

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 

 

17

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

18

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 

19

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 

20

what is rapamycin?

a drug that inhibits mTOR activity - breaks tln initiation 

cancer drug b/c mTOR is often extremely active in cancer cells

21

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

22

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

 

23

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

24

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 

25

what is RNAi?

RNA interferenceRNA 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

26

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 

27

problems w/ siRNA as a pharmaceutical to treat disease?

problems w/ siRNA delivery, lifetime in vivo, and specificity trouble the field

28

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

29

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

30

where does miRNA regulation work?

developmental pathways for gene expression

Decks in MCG Class (77):