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genes and gene expression > Mechanism and regulation of translation II > Flashcards

Mechanism and regulation of translation II Flashcards

1
Q

43S pre-in. complex also interacts with 3’ end of mRNA via poly(A) tail, how?

A

by ability of poly(A) binding protein (PABP) to bind to both eIF4G and eIF4B that is part of 43S pre-in. complex.
results in circularisation of mRNA by association of 5’ end with the 3’ end

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2
Q

what are sucrose gradients?

A

fractioning of macromolecules in the presence of a sucrose solution.
sucrose is used because it’s cheap and soluble

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3
Q

how are ribosomal complexes separated in a sucrose gradient centrifugation?

A

40s at the top, complex size increases as you go down the test tube and polysomes at the bottom

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3
Q

what happens when initiation is inhibited?

A

polysomes become fewer and smaller and single 80S ribosomes accumulate

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4
Q

inihibition of eukaryotic translation; cycloheximide

A

inhibits eEF2-mediated tRNA translocation through binding to the E-site of the 60S ribosomal unit

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5
Q

inhibition of eukaryotic translation; diphtheria toxin

A

ADP- ribosylates host eEF2-2 and inactivates it

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6
Q

inhibition of eukaryotic translation; puromycin

A

causes premature chain termination by acting as an analogue of aminoacyl-tRNA

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7
Q

35S-Methionine assays

A

used to look at the rate of protein synthesis

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8
Q

how is 35S-Methionine used to look at rate of protein synthesis?

A
  • added to tissue culture media and incubated with cells for 1 hour (5% CO2 at 37 degrees)
  • after incubation and media is removed, cells washed and lysed using sodium hydroxide
  • protein precipitation and filtration carried out using 10% trichloroacetic acid
  • samples are filtered through Whatman glass fibre GF/C discs and subsequently placed in scintillation fluid
  • radioactivity of each sample counted in scinitillation counter to determine counts/min (cpm)
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9
Q

Western Blotting

A
  1. sample prep
  2. gel electrophoresis
  3. potein transfer
  4. blocking
  5. primary antibody incubation
  6. secondary antibody incubation
  7. protein detection and analysis
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10
Q

global regulation of translation

A
  • usually by modification of translation initiation factors
  • achieved by changes in phosphorylation state of these factors by regulators that interact with them
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10
Q

mRNA specific regulation

A

uses elements in the 5’ and 3’ untranslated region

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11
Q

regulatory factors involved in inhibition of protein synthesis

A
  • dephosphorylation of 4E-BP1 (tumor suppressor protein)
  • eIF4G cleavage by caspases
  • phosphoryation of eIF2a
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12
Q

eIF4E (oncogene)

A
  • overexpression of eIF4E leads to loss of cellular growth control
  • eIF4E is often overexpressed in tumors, where main method of initiation is CAP dependent
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13
Q

structure of polypeptide chain eIF2

A

three subunits;
- GTP binding site; on y subunit
- phosphorylation site; on a subunit, ser 51
- K boxes; on b subunit, involved in interaction of eIF2B and eIF5

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14
Q

what happens in eIF2B is not phosphorylated?

A

guanine nucleotide exchanges
- eIF2B is also known as gunaine exchange factor (GEF)

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15
Q

eIF2B functions

A
  • eIF2B is a key regulator of mRNA translation
  • responsible for recycling of eIF2 which is required to allow translation initiation to occur
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16
Q

eIF2B structure

A
  • multisubunit protein, five different subunits termed a-e in order of increasing size
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17
Q

what does the E subunit of eIF2B do?

A
  • promotes GDP release from y subunit of eIF2 followed by conformational change
  • allows transfer of GTP residing in pocket of eIF2B, another conformational change followed by subsequent dissociation
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18
Q

what does tighter binding of eIF2 to eIF2B on eIF2a phosphorylation do?

A

very likely to interfere with the conformational changes necessary for catalyses abrogating eIF2B function

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19
Q

what happens when eIF2a is phosphorylated?

A

guanine nucletotide exchange is blocked

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20
Q

what is phosphorylation of eIF2a promoted by?

A
  • nutrient starvation
  • cellular stresses including heat shoc and unfolded protein response
  • viral infections and interferons
  • some cytokines
  • treatments that induce apoptosis
21
Q

what is phosphorylation of eIF2a regulated by?

A

serine 51 of the alpha subunit

22
Q

what are the kinases that phosphorylate eIF2a?

A
  • the heme regulated inhibitor (HRI) also known Heme controlled repressor (HCR)
  • the double stranded RNA activated protein kinase (PKR)
  • the GCN2 gene product S-cerevisiae
  • the PKR-like endoplasmic reticulum kinase (PERK) known as PEK
23
Q

where was protein kinase R (PKR) first identified?

A

in rabbit reticulocyte lysates where it is constitutively present.
in other mammalian cells levels of PKR can be induced after interferon treatment

24
Q

what is PKR depndent on?

A

dsRNA for its activity

25
Q

what does PKR do?

A

active pKR phosphorylates the ser 51 residue on the alpha subunit of eIF2a = in inhibition of eIF2B and inhibition of protein synthesis

26
Q

what hapens when PKR dimerises?

A
  • dimerises and undergoes phosphorylation after dsRNA activation
  • during viral infection, transcription or replication of viral genomes can lead to production of dsRNA.
    these are thought to activate PKR as part of antiviral mechanisms
27
Q

so what leads to phosphorylation of eIF2a?

A

RNA binding, dimerisation, autophosphorylation and activation of PKR leads to phosphorylation of eIF2a

28
Q

what other kinases can phosphorylate eIF2a?

A
  • PKR-like ER kinase
  • PERK is an ER-transmembran kinase that can phosphorylates eIF2a
  • the ER is the site of protein folding
29
Q

ER stress conditions

A

depletion of calcium when the ER is flooded with excess protein
- viral infection such as HCV infection > activation of PERK > this triggers the unfolded protein response

30
Q

what are 3 stress sensors found on ER membrane?

A

IREa1, PERK, ATF4

31
Q

what happens in non stressed cells?

A

molecular chaperone BiP binds to IREa1, PERK, ATF4

32
Q

what happens in stressed cells?

A
  • BiP is released, binds to unfolded proteins and activates signalling cascades
  • leads to reduction in global protein synthesis
  • get a specific up-regulation of TFs
33
Q

what does activation of PERK lead to?

A
  • eFI2a phosphorylation
  • global protein synthesis is inhibited
  • selective translation of ATF4, ATF4 has regulatory sequences in the 5’UTR that require phosphorylation of eIF2a in order to be translated
  • ATF4 induces expression of UPR target genes, involved with aa biosynthesis and transport
34
Q

unfolded protein response

A

cellular stresses that inhibit protein secretion > accumulation of unfolded proteins in ER lumen > activation of PERK (eIF2a phosphorylation) >
1. inhibition of global proteinsynthesis OR
2. activation of Ire1a/ATF4 induction of UPR target genes and transcription of chaperone proteins > mRNA specific regulation

35
Q

eukaryotic translation intitiatior factor 4E-binding protein 1 (4E-BP1)

A
  • tumor suppressor protein
  • interacts with eIF4E to prevent CAP recognition and eIF4E/4G interaction
  • inactivated via phosphorylation by members of the PI3K/Akt pathway; mTOR
36
Q

what is the availability of eIF4E to bind to eIF4G regulated by?

A

4E-BPs

37
Q

process of interaction of 4E-BP1

A
  1. mTOR can phosphorylate 4E-BP1
  2. during cell stress 4E-BP1 is dephosphorylated and activated
  3. activated 4E-BP sequesters eIF4E from eIF4G
38
Q

4E-BP1 and eIF4G

A
  • 4E-BPs compete with eIF4G for binding to eIF4E
  • have similar aa sequence motifs that are recognised by eIF4E
39
Q

what does TRAIL treatment of Jurkat lymphoma cells cause?

A

inhibits protein synthesis bc of 4E-BP1 sequestering eIF4E from eIF4G

40
Q

eIF4E and transformation

A
  • normally eIF4E is expressed at low levels and is least abundant of the IFs
  • eIF4E is referred to as a potent oncogene, it is found to be over-expressed in tumors
  • eIF4E essential for CAP dependent translation
41
Q

mRNA and transformation

A
  • CAP bearing mRNAs compete for eIF4E
  • mRNA that are required for the translation of proteins involved in tumorigenesis e.g. c-MYC, VEGF etc described as weak messages
42
Q

what do weak messages contain?

A

high degree of secondary structure in the 5’UTR of the mRNA

43
Q

what is eIF4E a requirement by?

A

GC rich mRNAs/ highly structured mRNAs

44
Q

GC rich mRNAs/highly structured mRNAs

A
  • less efficiently translated that strong ones
45
Q

what is excessive eIF4E due to?

A

the over-efficient translation of key growth regulatory mRNAs that contain extensive secondary strucuture in their 5’ ends

46
Q

what does the secondary structure on 5’ ends do?

A

normally, restrain translation of key growth regulatroy mRNAs but its effects are overcome by higher concentrations of eIF4E-containing complexes (as this includes eIF4A)

47
Q

what can overexpression of eIF4A lead to?

A

more efficient translation of these GC rich messages e.g growth promoting gene products, VEGF, cyclin D1 and c-MYC

48
Q

high eIF4E and low 4E-BPs

A

high eIF4E critical for translation of mRNAs encoding growth promoting and anti-apoptotic proteins

49
Q

low eIF4E and high 4E-BPs

A

low eIF4E may favour translation of mRNAs encoding growth inhibitory and pro-apoptotics proteins

50
Q

cell signalling in translation

A
  • high energy consuming process that is tightly regulated
  • signal transduction cascade respond to extracellular and intracellular cues to phosphorylate proteins involved in translation
  • phosphorylation events can regulate translation of both specific and total mRNAs
  • alterations in regulation= dysfunction and disease
  • many signalling pathways are involved, mTOR pathway is a key player
51
Q

what is phosphorylation of 4E-BPs regulated by?

A

a wide range of physiological stimuli

52
Q

mTOR

A
  • a central nutrient sensor that signals a cell to grow and proliferate
  • 4E-BP1 activity thought to be regulated by mTOR dependent phosphorylation
  • mTOR activity regulated by growth factors and aa availibility as well as energy status of the cell

genes and gene expression (14 decks)

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