Euk translation regulation (mRNA)

Question 1

what are the main steps in translation in euk?

Answer 1

initiation
elongation
termination

Question 2

what is translation?

Answer 2

synthesis of long chains of amino acids from a mRNA transcript using a ribosomes

Question 3

describe a ribosome

Answer 3

they are large multisubunit complexes (ribonuceloproteins)

Question 4

what are eIF?

Answer 4

eukaryotic initiation factors are proteins that can promotoe translation and transcription initiation

Question 5

why is translation initation important?

Answer 5

Rate limiting step of translation so
the bulk of translation regulation occurs at the initiation phase

Question 6

what are the steps of translation intiation in euk?

Answer 6

ribosome neeeds to be prepared

ribosomal entry point needs to be found

the start codon needs to be found

Question 7

why is the 5’ m-G cap important in translation intiation

Answer 7

enables the capping binding complex to be formed which helps the ribosome align and dock onto the mRNA properly

Question 8

how does ribosomal scanning work?

Answer 8

scanning model of initiation occurs so that the start codon can be found and elongation can begin

ribosome uses eiF4A/B helicase activity to unwind the mRNA secondary structure

Question 9

what is the function of Kozak sequence?

Answer 9

the AUG start codon should best match the Kozak which helps with ribosomal recognition of the AUG

then allows 48S complex to form

Question 10

before forming the 43 pre-initiation complex (40S+ternary complex), the 40S ribosome binds to other eIFs. Why is this?

Answer 10

binds to eIF1, 1A and 3

> this helps to seperate the 60S and 40S subunits and prevents reassociation of them until after the initiation codon has been found (scanning)

Question 11

how is the 60S ribosomal subunit recruited

Answer 11

once 48S complex is formed, the eIF sneed to be released via GTP hydrolysis

then 60S can associate with 40S creating 80S initation complex

Question 12

what 3 main things can influence translation initiation efficiency?
in Euk

Answer 12

5’ cap - if no there then complete absecne of tranlation

RNA hairpins - these riboswitches can pair strongly and block helicase activity

AUG - if strong or weak concensus can affect rate of synthesis

Question 13

why are pico-viruses cool?

Answer 13

they are single.stranded RNA viruses and do not have a 5’ cap and contain several strong AUG codons

yet they can be translated!

Question 14

what did picovirus research inform us about

Answer 14

it told us that ‘‘cap-Independent’’ translation initiation is another mechanism of tranlation :0

Question 15

how does cap- independent translation intiation work?

Answer 15

they use IRES to recruit ribosome internally instead of having a 5’cap
(particualarly useful in pro with multiple ORF)

Question 16

do Euk use cap-independent mechanism of translation?

Answer 16

yes!
often in times when cap-dependent is insufficient/ global translation is downregulated
e.g during mitosis

Question 17

name some examples of when cap - independent translation would be used?

Answer 17

oncogene proteins
growth factors
apoptopsis process
(during mitosis)
hypoxia, heat stress, ox stress
(during cell stress)

Question 18

how did we discover that cap - independent mechanism happened?

Experimental method

Answer 18

use a bicistronic construct using luciferase as a reporter.
> BI do 2 genes in tandem with stop codons

luciferase does not have 5’cap and instead under control of an IRES

Question 19

what specific processes is cap-independent translation important for in animals?

Answer 19

very important for patterning and development of fly and mice

IRES acts as an extra layer of gene regulation

Question 20

describe the closed loop model

Answer 20

model suggests that RNA is translated in a non-linear way
3’ interact with 5’ end

as ribosome in close proximity to 5’cap, reinitation is much quicker/efficient and ribosome is recycled

Question 21

why is the transcriptome bigger than proteome?

Answer 21

there can be many PTMs increasing the diversity of mRNA transcripts produced

but not all become functional proteins

Question 22

name 3 ways that translation can be globally downregulated in euk?

Answer 22

prevent ternary complex from forming which block the 48S formation

block capping binding complex forming

Question 23

what is the main way/process that translation downregulation is controlled?
what molecules are used?

Answer 23

phosphorylation by kinases which influences binding and release of different eiF and e-BPs

Question 24

when would translation downregulation be used?

Answer 24

during cell stress, stress granules appear in cytoplasm

they contain transcriptionally silent / stalled mRNAs allowing cell to respond to stress

Question 25

what a p-bodies?

Answer 25

cytoplasmic foci of mRNA degradation, always present in cytoplasm

Question 26

what is eiF2B? what is it role?

Answer 26

this is the guanine exchange factor for eIF2 it promotes GTP-GDP exchange and reactivation of ternary complex

Question 27

how is eiF2B activity regulated

Answer 27

if eiF2 is *phosphorlyated* then 2B can't bind and activate ternary complex so translation downregulated

Question 28

why would blocking eif4E downregulated translation in euk?

Answer 28

prevents formation of capping -binding complex which is NECCESSARY FOR translation initiaiton

Question 29

how does eiF4E regulate translation downregulation?

Answer 29

eiF4E can bind to the extended n-terminal of 4G but this interaction can be blocked by different eiF4E- BP which also have a concensus 4E binding site

Question 30

what features do cancer cells have that can promote survival + proliferaiton?

Answer 30

there is hyperactivation of the cap-dependent pathway of translation intiaiton// there is no global downregulation as they have hyperactivation of GF signalling pathways which results in 4E-BP phosphorylaiton enabling translation to take place *uncontrolled translation NOT downregulated*

Question 31

how can mRNA translation be LOCALLY regulated?

Answer 31

the UTRs are key regions to determine stability, localisation and translation of mRNA as they are sites where RBP and microRNAs can bind

Question 32

how do microRNAs influence local translation of mRNA?

Answer 32

they act to degrade mRNA completely or inhibit translation depending on the amount of complementarity they have using the Risk complex

Question 33

how can RBPs influcence local translation regulation?

Answer 33

they can recruit ei4E-BP or contain that concensus motif themself so interact with eif4E directly or indirectly and can block translation happening

Question 34

how can RBP translation regulation be overcome?

Answer 34

by phosphorlating the mRNA, polyApolymerase and BP can be recruited and eiF4G can outcompete the RNA-BP allowing translation to happen by forming closed loop model

Question 35

in times of low iron, how can translation of transferrin receptor be altered?

Answer 35

RNA-BP (IRP) will occupy the 3'UTR of the transferrin receptor mRNA this stabilises it and allows it to be translated

Question 36

in times of low iron how would ferritin mRNA be regulated?

Answer 36

ferritin stores iron so we dont want it translated IRP will sterically block the 5'UTR of ferritin mRNA and prevent 43S complex to be recruited and so prevents ferritin translation

Question 37

in times of high iron how would transferrin receptor mRNA be regulated?

Answer 37

TransR will allow iron to enter cell - but don't want that IRP (the rna-bp) WONT occupy 3' UTR of transferrin receptor mRNA susceptible to degradation by endonuclease/unstable