#28 Gene Regulation 10.24.12 Flashcards Preview

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Lecture objectives

1. Describe how RNA editing, mRNA decay or by altering either the start site, splicing or 3" processing affets teh protein product

2. Describe the different strategies of translational control (pay attention to molecular basis by which RIBOSWITCHES< IRESs, miRNAs and RNAis work)

3. Describe th role of miR-21 in cancer

2

What atre the 6 sites for gene expression control>

Transcriptional control

RNA processing conrol

RNA transport and localization control

Translation Control (

mRNA degradation control

Protein activity control (covalent modification or by complex formation with another protein or small ligand)

3

What kind of steps are invovled in gene expresison

Transcription, pre-mRNA processing, nuclear export, translation, and in some ases protein activiation (phosphoryation)

4

what are long noncoding RNA (lncRNA)? how many more lncmRNA are transcribed than mRNA? What is the funciton?

>200 nts long

10-20x more of the nts in the genome are transcribed as lncRNA than as mRNA

Expected to play a KEY ROLE in regualtion of gene expression

5

What is the best studied example of long noncoding mRNA and what is its function

Xist lncRNA

essential for inativation of one copy of the X chromosome in females

This lncRNA "coats" this chromosome
Recruits histone remodeling complex
Trimethyates specific histone lysine
Silent transcription

6

What happens if you didn't silence one copy of X in females?

dose issue

7

What is the point of "Leaky scanning" ( variety of either using first AUG or later AUG as start site) to initiate translation

allows there to be a control of signal sequences for the protein ,providing control of where in teh cell a protein is located

8

How is guanine nucletoide ehsange factor employed in normal translation

Guanine nucleotide exchange factor (eIF2B) recycles GDP in eIF2 after translocation. This exhcange saps GDP for GTP, transforming inative eIF2 to active form!

9

How is "leaky scanning" employed during STARVATION

during amino acid starvation, protein kinase Phosphorylates the GDP formm of eIF2-->becomes trapped in an INACTIVE COMPLEX with eIF2B

This dramaticaly SLOWS down tranlsation ,thereby increasing the likelihood that a later AUG will be used for translational initiation

10

what does phosphorylation of GDP-bund eIF2 (inactive) do?

It traps eIF2in an inactive complex

11

What is the purpose of alternate splicing?

it allows for many different proteins to be translated form one gene

12

What kind of proteins control alternate splicing?

RNA binding proteins

13

How does negative control regulate alternative splicing?

repressor molecule binds to a splice junction. Binding of repressor portins sometimes uries the splice site, maksing it so that splicing mahcinery doens't recognize th splice site

14

How does positive control regualte alternative splicing

bindign of splicing activotro to an ENHANCER ELEMENT can recruit th splciing machiney to a poorly defiend splice site

15

Why are elements called "enhancers"

they can bind far from splice site that they are regulating

16

How can alternate 3' cleavage of the message lead to two different forms of an anitbody?

one that is secreted and anotehr that is membrane bound

17

Describe the membrane bound antibod formation

Membrane bound contains C-terminal Hphobic region and occurs in unstibmulated B lymphocytes

inron containing a PREMATURE STOP CODON has been spliced out and the full length protein is made (Antibody bound to membrane)

18

Describe formation of stimulated B lymphocyte that are not bound to membrane

Expression is incresed for a subunit of FstF (cleavage stimulator factor).

As a result of this higher concetnration, an earlier 3' cleavge site on the pre-mRNA is recognied and it is cleaved inside the intron and then polyadenylated.

This second form of hte antibod is shorter and b/c it is missing the hphobic tail f the full-lenght protein, it is secreted

19

What is CstF and function?

it is cleavage stimulator factor

Higher concentration of cleavage stimulator factor leads to an earlier3 cleavage site on the pre-mRNA is recognized and is cleaved INSIDE the INTRON and then polyadenylated

2nd form of antibody is shorter and b/c it is shorter and b/c it is missing the Hphobic tail of the full-length protein

20

What is RNA editing? When does it happen

process that occurs after transcription and thus modifies the resulting translated protein sequence

ADAR's chnage A--> I

There can also b editing in splicing

21

What are ADARs?

Adenosome Deaminases acting on RNAs-

estimated to carry our A--> I (adenosien to inosine) editing in over 1,000 different human genes

22

How does A--> I change the translated protein? What is the complementary base of I?

A usually pairs with U

I is like G and pairs with C

I pairs with C

23

Does ADAR recognize ss or ds RNA?

DS RA to b editted and its complement

The edited sequenc eis foten int ehe xon region with the complementoary region in the intron, near the 3' inron jucntion

24

There is a transmitter gated ion channel in teh brain that changes A to I. TWht does this do>

it causes Gln--> Arg mutation ,that alterst ehh claciumpermeability in the channel

25

What type of RNA editing is involvd with changing C-->U (in liver>)

What aobut in intestine? for ApoB-28

C is daminated to U

releavant example with two forms of ApoB protein

in the liver ApoB-100 is an essential component of chylomicrons and VLDL (NO EDITING..it is CAA)

Om omtestome. CAA Gln codon is edited to a UAA stop codon, resulting in shorter ApoB-48 protein that incorporates into chylomicrons

26

What does C--> U deamination do in liver?

ApoB is an essential component of chyomicrons and VLDL

27

What does RNA editing do in the instestine, hwne it chages from CAA ocdon to UAA

resultsin a shorter protein that incorporates into chylomicrons

28

Which mRNA is more stable? Bacterial or Eukaryotic?

Bacterial . Most bacterial mRNA is made quickly and degraded quicky, allowing these simple orgnaism to adapt rapidly to their ever changing environment . A typical bacterial mRNA has a half-life of about a minute or two

Euk mRNAas halve a typical half life of 3- minutes but soem last up tot 10 hours

29

How many nucleotides at the tail ends until degradation beins at both end?

25 nucleotides in humans

3'Poly A tail is gradually shortened

5' Cap (decapping)

30

What happens once decapping has taken place

Rapid 5' -->3' RNA degradation occurs. Likewise rapid degradation occurs at the 3' end

31

What is Deadenylase?

binds the cap at the 5' end of the transcript and degrades Poly A tails at the 3'end (looks like pacman)

32

Is there compettion between translation initaiton and mRNA decay? What proteins/molecules rae invovled?

Yes!
At initiiation, eIF4G recognizes both 5' and 3' features

33

What kind of modiications to SDS (shine-Dalgarno seqeunce) can be done in for Translational control in Porkayrotes?

Translational repressor protein buries the SDS

Temperature dependent stem loop buries SDS (i.e.i Listeria monocytogenes that only allow SDS and its expression inside the warm body)

Riboswitch --2 didfernet ocnformations
(small molecule that either stabilizes by bidnign and shuts off translation by burying SdS in stem-loop structure) or not

Antisense RNA sued to bury SDS sequence- bacteria employ this method in iron -storage genes

34

Expalin the Translation repressor protin in bacterial translation control

Binding of trnalsational repressor protein buries the SDS, inhibiting initiation of translation

35

Explain tempearture dependet stem loop and translational regualtion

Stem loop buies SDS

only at elevated temperatures doesloop open up and permit initiation of trnalsation

Example is Listeria monocytogenes (bacteri that doesnt unfold and expose SDS until it is inside the body

36

Explain 5'UTR folds that have 2 different conformations and translational regulation in relation to RIBOSWITCHES

one conformation, stabilized by binding of a small moleucle, shuts off tranlsation by burign SDS in a stem-loop structure

TOher conformation occurs int eh absence of the small moleucl, prmostes an accesible SDS conformatino taht in turn promotes tranlation

37

How does antisense RNA affect translational regualtions

antisense strand buries SDS sequence

Bacteria emplys this strategy to regulate iron-storage genes

38

What are Riboswithces? Can they control transcription or translation or both?

Can control both transcription and translation

39

Are riboswithces more numerous in Euk or Prok? What kind in Euk?

More extensively in bacteria!

Thiamine pyrophosphate is the only one foudn in eukartyoes

40

Describe riboswitch mode of action in transcriptional control

the 5'UTR contains all teh necessary information in a riboswitch

under conditions of excess guanine, base binds to riboswitch, stabiliizng a conformation of the riboswitch that contains a transcription terminator --> inhibits transcription (OFF)

Under conditions of insufficient guanine--> base dissociates from riboswitch, now adopts a conformation that buries the terminator elemetn and thereby allos transcription to occur (ON)

41

What is transferrin

soluble protein that carries iron in the blood; needed when low iron (iron starvation) to bring into cells

42

What is ferritin?

assembly of 24 subunits used to store itnracellular iron in a form that is not toxic to the cell and is needed under excess iron conditions; activiated when too `much iron

43

How does aconitase lease to transferrin receptor being made and importing of iron bound to transferrin? (under starved iron condtions)

Aonitase has an RNA bindign domain that recognizes and binds to a stem-loop structure int eh 3' UTR of the transferrin receptor mRNA

This binding of aconitase to the mRNA blocks an endonucleolytic cleavage site in mRNA (which would otherwise lead to ENDOnucleoltyic mRNA decay if cleaved)

44

How does aconitase shut off translation of ferritin during Iron starvation?

aconitase binds to a similar stem loop structure in teh 5'UTR of the ferritin mRNA to block INITIATION of TRANSLATION

45

How does acontinase allow the creation of Ferrtin during excess iron conditions

Free iron is toxic to a cell

Free excess iron binds to aconitase, which in turn dissociates form teh stem loop structure in teh 5'UTR of ferritin mRNA to permit initiation of translation

46

How does aconitase inactivate translation of transferrin mRNA

excess iron binds to aconitase, chanign its conformatino and causing it to dissocaite from RNA stem loop structure in teh 3' UTR

Dissociation of aconitase exposes endonucleoltic cleavage site int eh mRNA , which in turn is cut to ttirgger mRNA decay that prevents tranlsation

47

What does IRESs stand for?

Inernal Ribosome Entry Site -

CAP independent translational intitiation

48

Where can IRES occur?

occurs anywhere within the gene, sometimes leading to the production of two completely different proteins

49

What is required for IRES to function?

Diffferent proteins are invovled in IRES intitiation and translation and also note that it requires that IRES RNA is folded

50

How do viruses exploit host IRESs to force host to stop expressing its genome and express the viral genome instead?

Virus disrupts interaction between CAP complex and PolyA Binding Protein by releasing a site-specific protease that selectively truncates eIF4G

51

What happens with truncated eIF4G'?

no longer has abilti to bind the CAP complex, but is still able to bring two ends of message together by simultaneously binding to the IRES on one side and the polyA tail via the PAP on the otehr side

52

what are miRNAs? Funciton?

microRNAs-

play important role in
cell development,
cell differentiation,
cell cycle regulation, apoptosis

53

What is the role of miRNA is cancer?

studies show that miRNA is dereglated in cancer
miRNAs can act as tumor suppresors and oncogenes

30% of human genes are regulated by miRNA

54

What transcribes miRNA?

Pol II transcribes the often long miRNA transcripts (pri-miRNAs)

55

What recognizes and cleaves this primary transcript (pre-miRNA?_

endoribonuclease DROSHA --> to form precursoer pre-iRNA

56

What exports pre-miRNA into cytoplams?

Exportin 5 into the cytoplasm wher eit is furtehr processed

57

What is DICER?

endonuclease Dicer- processes it into an miRNA duplex

58

what is RISC?

RNA-induced silencing complex

only one of the two strands of the miRNA assembles with a RISC . Tehre are two outcomes

59

What are the two possible outcomes after the binding of one strand of miRNA with RISC

1. is single strand of miRNA in RISC complex extensively mathces the target mRNA, target MRNA will be cleaved y ENDONUCLEASE ARGONAUTE

60

--> INTERNATL CUTTING OF THE MRNA LEADS IN TURN TO TMRNA DEGRADATION

IF SINGLE STRAND OF MIRNA IS RISK COMPLEX doesnt extensively match the target mRNA , TRANSLATION IS INHIBITED and the mRNA is destailied by shortening the poly A tail

mRNAs are targeted to processing bodies (P-bodies) for eventual degradation

61

What are RNAis?

Invovled with translational or transcriptional repression

involdved in defense mechanism that destroys foreign RNA

62

how do double stranded RNA in the cell (viruses) trigger RNAi?

by recrutiing the protein complex Dicer , which cleaves the RNA into ~23 nucleotide dubplex fragments called small interfering RNAs (siRNAs)

63

Once cleaved into siRNAs and bound with argonaute, what are the two possible fates >

this RNA-protein complex (RNP) can form a RISC complex --> leading to TRANSLATIONAL INHIBITION of an mRNA with complementary seqence. RISC RNA complement to teh rforeign RNA si a perfect match, which thsu triggers degradation of foreign cellular RNA conaminant

2. Argonaute-siRNA RNP can selectively shut off syntehsis of target RNAs. argonaute-siRNP assembles with RNA-induced transcriptional silencing RITS complex

64

What is a RITS complex

RNA Induced Transcripational Silencing

single stranded siRNA acts as a guide to bind complelemtnary RNA transcripts that emerge from Pol II

RITS complex recruits enzymes to modify histones and DNA (Methylation etc), thereby silencing this region of chromatin

65

How are RNAi powerful biochemical tool?

use dto knowck down expression of any desired mRNA