LEC27: Translation Mechanism II & Regulation

Question 1

what are the steps of translation elongation?

Answer 1

1) tRNA anticodon:codon interaction
2) peptidyl transferase rxn, aka peptide bond formation
3) translocation

Question 2

what is “dwell” time? its effect?

Answer 2

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

Question 3

what is eEF1A? what does it do?

Answer 3

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

Question 4

what helps hydrolysis of eEF1A occur?

Answer 4

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

Question 5

what is basis of specificity in a.a. protein synthesis?

Answer 5

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

Question 6

how many high energy bonds are hydrolyzed during polypeptide chain synthesis?

Answer 6

4 high energy bonds are hydrolyzed:

2 in aa-tRNA synthetase rxn, 2 in elongation rxn

Question 7

what is translocation?

Answer 7

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

Question 8

what catalyzes translocation?

Answer 8

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

Question 9

what are stop codons?

what recognizes them?

Answer 9

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

Question 10

what is peptide tunnel?

Answer 10

where growing polypeptide chain emerges during process of elongation

emerges N-terminus first

Question 11

what’s a polysome?

Answer 11

an mRNA that has > 1 ribosome translateing it

useful to isolate v. actively translated mRNAs

Question 12

how do 3’ - 5’ mRNA ends’ interactions occur? what is result?

Answer 12

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”

Question 13

are bacterial and human ribosomes the same?

Answer 13

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

Question 14

at what levels can tln be regulated?

Answer 14

1) globally
2) mRNA-specific regulation

Question 15

why is tln energetically expensive?

Answer 15

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)

Question 16

how can translation be globally inhibited?

describe mechanism

Answer 16

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 **

Question 17

dsecribe global inhibition of translation via stress signaling

Answer 17

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

Question 18

describe global activation of translation initiation

Answer 18

**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 **

Question 19

what is mTOR

Answer 19

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 **

Question 20

what is rapamycin?

Answer 20

a drug that inhibits mTOR activity - breaks tln initiation

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

Question 21

how does **specific inhibition of mRNA **occur?

Answer 21

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

Question 22

what is ferritin? how is ferritin expression regulated?

Answer 22

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

Question 23

what happens if regulatory protein binds in 3’ UTR?

Answer 23

endonuclease is recruited that cleaves message, exposes a 3’ end for rapid degradation by 3’ endonucleases

method of mRNA-specific regulation

Question 24

what is miRNA?

how does mechanism work?

Answer 24

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 **

Question 25

what is RNAi?

Answer 25

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

Question 26

how can one do “knock down” of expression of a target gene?

Answer 26

introduce siRNA (small interfering RNA)

targeted knock-down gene expression

potential pharma took to treat disease

Question 27

problems w/ siRNA as a pharmaceutical to treat disease?

Answer 27

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

Question 28

what is drosha?

what happens after it acts?

Answer 28

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

Question 29

what is RISC?

Answer 29

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

Question 30

where does miRNA regulation work?

Answer 30

developmental pathways for gene expression