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27 Cards in this Set
- Front
- Back
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when does "commitment" occur in intron splicing?
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when a splice site has been targeted by a sequence specific RNA BP, which induces spiceosome assembly
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What proteins often serve to commit splicing of introns? What is unique about them?
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SR proteins, rich in Arg/Ser
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What types of RNA can SR proteins bind?
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specific intronic regions and splicing enhancers
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What types of trans-acting factors participate in pre-mRNA splicing?
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snRNPs (U1,2,4,5,6)
3' splice site selectors (Slu 7, U2AF) SR proteins (SC35, SF2, ASF) splicing regulatory proteins (Tra2, Sam68) |
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How was is discovered that U1 defines the 5' splice junction by base complementarity?
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mutate mRNA so RNA/U1 cannot bind--> no splicing
restore bp by mutating both mRNA and U1 so they DO bind and restored splcing ** bp is necessary for splicing but not sufficient |
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How are most interactions between snRNPs mediated?
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primarily through formation of bp but secondarily through protein/protein stability in some complexes
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What is role of U6? Where does it bind?
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-replaces U1 in active spliceosome
-U6 binds to 5' end of intron |
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What is the role of U5 in spliceosome? What experiment was done to uncover this?
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U5 brings together the 5' and 3' end of intron by positioning itself close to 3' end of E1 and 5' end of E2
Crosslinking studies were done to conclude this |
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Describe 3 ways SR proteins contribute to the regulation of splicing.
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1. mediator between 3' site selectors and U1 snRNP
2. strengthening weak 3' sites by binding exonic splicing enhancer sequences 3. regulating splice site selection by competing with splicing inhibitors |
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What are site selectors?
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proteins that bind to specific splice acceptor sites (Slu 7, U2AF)
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How is inclusion of exon 5 of CD44 repressed?
How is it reversed? |
1. hnRNP A1 binds the exonic splicing silencer region on RNA, which inhibits U2 and U2AF (which bind intronic sequences) and TRA2, ASF/SF2 (which bind exonic splicing enhancer)
2. Ras* ---> ERK*--->P-Sam68 Sam68 inhibits hRNP A1 via competing with ESS or inhib while both proteins on ESS |
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1.What are Group I and II introns?
2. How discovered? 3. How do Group I and II introns excised? |
1. self splicing genes (ex.catalytic RNA)
2. some genes code for r/t/mRNA and no protein or ATP needed 3. Group I: transesterification rxn initiated by G Group II: internal branch site involving A |
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1. What are ribozymes?
2. What are their main rxns? 3. What is guide sequence? 4. All ribozymes must have ___ for activity 5. Ribozymes can also bind ____ |
1. RNA molecules capable of catalyzing rxns
2. RNA transesterification, RNA cleavage (hydrolysis of phosphodiester bonds) 3. Sequence on ribozyme that makes sure substrate is aligned into active site 4. 3D structure 5. metal ions |
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3 functions of mRNA 5' cap
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1. Transport through interaction with nuclear cap binding proteins
2. increases efficacy of translation by targeting formation of pre-initiation complex 3. protects transcript from 5--3 exoribonucleolytic activity ** very E rich rxn- very important |
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How is mRNA treated at 3' end?
Describe 3' mRNA processing |
poly(A) tail
-enzyme recog polyA signal (AAUAAA) and GU rich sequence 20-40nt downstream - endonuclease cleaves the transcript 10-30nt down from AAUAAA - ~200A added to 3' end using polyA polymerase |
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What is poly A bound to?
What does this interaction do? (2 functions) |
poly A binding proteins
1. Increase translation efficiency by complexing with eIF4G in pre-initiation complex 2. Protects transcript from 3-->5 exonucleases |
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Describe 2 ways to get multiple RNAs from 1 gene
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1. alternate poly (A) cleavage sites on DNA
2. alternative splicing |
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what role does poly(A) play in bacteria?
How was this discovered? How is this different from euk? |
degrades RNA
stable stem loop, blunt 3' end add degradeosome proteins wt--> no effect add A--> over time, RNA degrades Also if add ATP, increased degradation, so in addition to exonuclease must also be helicase in euk, polyA stabilizes mRNA |
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What is role of helicase in prok RNA degradation?
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unwind the stem, once helicase and exonuclease bind RNA
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What protein mediates mRNA processing and degradation in euk? What does it do?
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Mtr4 helicase
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Describe role of rrp44
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-rrp44 associates with exosome
-active nuclease -endonuclease activ -has RNA binding domain has catalytic site for exo activity - thought that RNA passes through pore of exosome to rrp44 where it is degraded |
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Describe role of rrp6
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- exosome independent
-active nuclease in NTD -can bind rrp47 - not sure if RNA pass through exosome first or if goes directly to rrp6 (but it can act independently) |
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What protein comprise the exonuclease degradation machinery?
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rrp44 and rrp6
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Describe role of mtr4
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- RNA helicase
- required for processing rRNA, U4 snRNA, snoRNA -degrades aberrant mRNA, tRNA, CUTs - helps make rRNA - KO is lethal - knockdown impairs ribesome biogenesis - helicase mutation impair processing and degradation |
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Describe proposed model for how RNA degraded via mtr4 and exosome
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- RNA target for processing
- exosome chews 3' end, gets stuck - Mtr4 assists in removes secondary structure - processing: mature RNA abberent RNA: degrade |
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Describe experiment to suggest that RNA degradation is promoted by polyA
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model: hypermethylated tRNA b/c initiated methionine not Me, slow growth phenotype
-cant' degrade hypermeth Met tRNA w/o polyA pol, mtr4, TRAMP complex **ATP not required |
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What is TRAMP?
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- 4 proteins:
1. trf4/trf5 (non-canonical RNA polymerase, add ~15-30 As to 3') 2. Air1, Air2 (Zn knuckle protein assoc trf4 to enhance) 3. Mtr4 4. polyA complex - adds A to 3' to enhance degradation - could be several rounds of A, exosome degrades somewhat, but TRAMP + exo shows significant deg ATED: TRAMP+exosome--> degradation |
