#28 Gene Regulation 10.24.12 Flashcards
Lecture objectives
- Describe how RNA editing, mRNA decay or by altering either the start site, splicing or 3” processing affets teh protein product
- Describe the different strategies of translational control (pay attention to molecular basis by which RIBOSWITCHES< IRESs, miRNAs and RNAis work)
- Describe th role of miR-21 in cancer
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)
What kind of steps are invovled in gene expresison
Transcription, pre-mRNA processing, nuclear export, translation, and in some ases protein activiation (phosphoryation)
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
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
What happens if you didn’t silence one copy of X in females?
dose issue
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
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!
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
what does phosphorylation of GDP-bund eIF2 (inactive) do?
It traps eIF2in an inactive complex
What is the purpose of alternate splicing?
it allows for many different proteins to be translated form one gene
What kind of proteins control alternate splicing?
RNA binding proteins
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
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
Why are elements called “enhancers”
they can bind far from splice site that they are regulating
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
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)
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
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
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
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
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
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
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
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
What does C–> U deamination do in liver?
ApoB is an essential component of chyomicrons and VLDL
What does RNA editing do in the instestine, hwne it chages from CAA ocdon to UAA
resultsin a shorter protein that incorporates into chylomicrons
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
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)
What happens once decapping has taken place
Rapid 5’ –>3’ RNA degradation occurs. Likewise rapid degradation occurs at the 3’ end
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)
Is there compettion between translation initaiton and mRNA decay? What proteins/molecules rae invovled?
Yes!
At initiiation, eIF4G recognizes both 5’ and 3’ features
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
Expalin the Translation repressor protin in bacterial translation control
Binding of trnalsational repressor protein buries the SDS, inhibiting initiation of translation
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
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
How does antisense RNA affect translational regualtions
antisense strand buries SDS sequence
Bacteria emplys this strategy to regulate iron-storage genes
What are Riboswithces? Can they control transcription or translation or both?
Can control both transcription and translation
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
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)
What is transferrin
soluble protein that carries iron in the blood; needed when low iron (iron starvation) to bring into cells
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
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)
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
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
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
What does IRESs stand for?
Inernal Ribosome Entry Site -
CAP independent translational intitiation
Where can IRES occur?
occurs anywhere within the gene, sometimes leading to the production of two completely different proteins
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
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
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
what are miRNAs? Funciton?
microRNAs-
play important role in
cell development,
cell differentiation,
cell cycle regulation, apoptosis
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
What transcribes miRNA?
Pol II transcribes the often long miRNA transcripts (pri-miRNAs)
What recognizes and cleaves this primary transcript (pre-miRNA?_
endoribonuclease DROSHA –> to form precursoer pre-iRNA
What exports pre-miRNA into cytoplams?
Exportin 5 into the cytoplasm wher eit is furtehr processed
What is DICER?
endonuclease Dicer- processes it into an miRNA duplex
what is RISC?
RNA-induced silencing complex
only one of the two strands of the miRNA assembles with a RISC . Tehre are two outcomes
What are the two possible outcomes after the binding of one strand of miRNA with RISC
- is single strand of miRNA in RISC complex extensively mathces the target mRNA, target MRNA will be cleaved y ENDONUCLEASE ARGONAUTE
–> 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
What are RNAis?
Invovled with translational or transcriptional repression
involdved in defense mechanism that destroys foreign RNA
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)
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
- Argonaute-siRNA RNP can selectively shut off syntehsis of target RNAs. argonaute-siRNP assembles with RNA-induced transcriptional silencing RITS complex
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
How are RNAi powerful biochemical tool?
use dto knowck down expression of any desired mRNA
