Mechanism and regulation of translation II Flashcards
43S pre-in. complex also interacts with 3’ end of mRNA via poly(A) tail, how?
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
what are sucrose gradients?
fractioning of macromolecules in the presence of a sucrose solution.
sucrose is used because it’s cheap and soluble
how are ribosomal complexes separated in a sucrose gradient centrifugation?
40s at the top, complex size increases as you go down the test tube and polysomes at the bottom
what happens when initiation is inhibited?
polysomes become fewer and smaller and single 80S ribosomes accumulate
inihibition of eukaryotic translation; cycloheximide
inhibits eEF2-mediated tRNA translocation through binding to the E-site of the 60S ribosomal unit
inhibition of eukaryotic translation; diphtheria toxin
ADP- ribosylates host eEF2-2 and inactivates it
inhibition of eukaryotic translation; puromycin
causes premature chain termination by acting as an analogue of aminoacyl-tRNA
35S-Methionine assays
used to look at the rate of protein synthesis
how is 35S-Methionine used to look at rate of protein synthesis?
- 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)
Western Blotting
- sample prep
- gel electrophoresis
- potein transfer
- blocking
- primary antibody incubation
- secondary antibody incubation
- protein detection and analysis
global regulation of translation
- usually by modification of translation initiation factors
- achieved by changes in phosphorylation state of these factors by regulators that interact with them
mRNA specific regulation
uses elements in the 5’ and 3’ untranslated region
regulatory factors involved in inhibition of protein synthesis
- dephosphorylation of 4E-BP1 (tumor suppressor protein)
- eIF4G cleavage by caspases
- phosphoryation of eIF2a
eIF4E (oncogene)
- overexpression of eIF4E leads to loss of cellular growth control
- eIF4E is often overexpressed in tumors, where main method of initiation is CAP dependent
structure of polypeptide chain eIF2
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
what happens in eIF2B is not phosphorylated?
guanine nucleotide exchanges
- eIF2B is also known as gunaine exchange factor (GEF)
eIF2B functions
- eIF2B is a key regulator of mRNA translation
- responsible for recycling of eIF2 which is required to allow translation initiation to occur
eIF2B structure
- multisubunit protein, five different subunits termed a-e in order of increasing size
what does the E subunit of eIF2B do?
- 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
what does tighter binding of eIF2 to eIF2B on eIF2a phosphorylation do?
very likely to interfere with the conformational changes necessary for catalyses abrogating eIF2B function
what happens when eIF2a is phosphorylated?
guanine nucletotide exchange is blocked
what is phosphorylation of eIF2a promoted by?
- nutrient starvation
- cellular stresses including heat shoc and unfolded protein response
- viral infections and interferons
- some cytokines
- treatments that induce apoptosis
what is phosphorylation of eIF2a regulated by?
serine 51 of the alpha subunit
what are the kinases that phosphorylate eIF2a?
- 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
where was protein kinase R (PKR) first identified?
in rabbit reticulocyte lysates where it is constitutively present.
in other mammalian cells levels of PKR can be induced after interferon treatment
what is PKR depndent on?
dsRNA for its activity
what does PKR do?
active pKR phosphorylates the ser 51 residue on the alpha subunit of eIF2a = in inhibition of eIF2B and inhibition of protein synthesis
what hapens when PKR dimerises?
- 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
so what leads to phosphorylation of eIF2a?
RNA binding, dimerisation, autophosphorylation and activation of PKR leads to phosphorylation of eIF2a
what other kinases can phosphorylate eIF2a?
- PKR-like ER kinase
- PERK is an ER-transmembran kinase that can phosphorylates eIF2a
- the ER is the site of protein folding
ER stress conditions
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
what are 3 stress sensors found on ER membrane?
IREa1, PERK, ATF4
what happens in non stressed cells?
molecular chaperone BiP binds to IREa1, PERK, ATF4
what happens in stressed cells?
- 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
what does activation of PERK lead to?
- 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
unfolded protein response
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
eukaryotic translation intitiatior factor 4E-binding protein 1 (4E-BP1)
- 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
what is the availability of eIF4E to bind to eIF4G regulated by?
4E-BPs
process of interaction of 4E-BP1
- mTOR can phosphorylate 4E-BP1
- during cell stress 4E-BP1 is dephosphorylated and activated
- activated 4E-BP sequesters eIF4E from eIF4G
4E-BP1 and eIF4G
- 4E-BPs compete with eIF4G for binding to eIF4E
- have similar aa sequence motifs that are recognised by eIF4E
what does TRAIL treatment of Jurkat lymphoma cells cause?
inhibits protein synthesis bc of 4E-BP1 sequestering eIF4E from eIF4G
eIF4E and transformation
- 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
mRNA and transformation
- 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
what do weak messages contain?
high degree of secondary structure in the 5’UTR of the mRNA
what is eIF4E a requirement by?
GC rich mRNAs/ highly structured mRNAs
GC rich mRNAs/highly structured mRNAs
- less efficiently translated that strong ones
what is excessive eIF4E due to?
the over-efficient translation of key growth regulatory mRNAs that contain extensive secondary strucuture in their 5’ ends
what does the secondary structure on 5’ ends do?
normally, restrain translation of key growth regulatroy mRNAs but its effects are overcome by higher concentrations of eIF4E-containing complexes (as this includes eIF4A)
what can overexpression of eIF4A lead to?
more efficient translation of these GC rich messages e.g growth promoting gene products, VEGF, cyclin D1 and c-MYC
high eIF4E and low 4E-BPs
high eIF4E critical for translation of mRNAs encoding growth promoting and anti-apoptotic proteins
low eIF4E and high 4E-BPs
low eIF4E may favour translation of mRNAs encoding growth inhibitory and pro-apoptotics proteins
cell signalling in translation
- 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
what is phosphorylation of 4E-BPs regulated by?
a wide range of physiological stimuli
mTOR
- 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
