Cytoplasmic regulation lifetime localization initiation
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Transcript of Cytoplasmic regulation lifetime localization initiation
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Cytoplasmic regulation
• lifetime
• localization
• initiation
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Post-transcriptional regulation1) mRNA processing2) export from nucleus3) mRNA degradation 4) mRNA localization5) initiation of translation varies >10x
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Initiation in Eukaryotes
1)eIF4E binds mRNA cap
• Won’t bind unless 5’cap is methylated
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Initiation in Eukaryotes
1)eIF4E binds mRNA cap
• Won’t bind unless 5’cap is methylated
• eIF4E & PABP protect RNA from degradation
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Initiation in Eukaryotes
1)eIF4E binds mRNA cap
• Won’t bind unless 5’cap is methylated
• eIF4E & PABP protect RNA from degradation
• eIF4E must be kinased to be active
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Initiation in Eukaryotes
1)eIF4E binds mRNA cap
• Won’t bind unless 5’cap is methylated
• eIF4E & PABP protect RNA from degradation
• eIF4E must be kinased to be active
• XS = cancer
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Initiation in Eukaryotes1) eIF4E binds mRNA cap2) eIF4G binds eIF4E, eIF4A & PAB: complex = eIF4F
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Initiation in Eukaryotes1) eIF4E binds mRNA cap2) eIF4G binds eIF4E, eIF4A & PAB: complex = eIF4F3) itRNA:met & eIF1, eIF2 & eIF3 bind 40S subunit
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Initiation in Eukaryotes3) itRNA:met & eIF1, eIF2 & eIF3 bind 40S subunit4) 43S complex binds eIF4F5) eIF4A & eIF4B scan down & melt mRNA (using ATP)
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Initiation in Eukaryotes3) itRNA:met & eIF1, eIF2 & eIF3 bind 40S subunit4) 43S complex binds eIF4F5) eIF4A & eIF4B scan down & melt mRNA (using ATP)6) 43S follows until finds Kozak sequence 5’-ACCAUG
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Initiation in Eukaryotes
7) initiator tRNA:met binds start codon (AUG), eIF2 hydrolyzes GTP
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Initiation in Eukaryotes8) 60S subunit binds
itRNA:met is at P siteribosome thinks it is a protein!why 60S subunit doesn’tbind until itRNA:met
binds AUGwhy have itRNA:met
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Regulation
1)Key step = eIF4E binds mRNA cap
• Won’t bind unless 5’cap is methylated
• eIF4E must be kinased to be active
• 4E-BP1 binds eIF4E & keeps it inactive until kinased
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Regulating Translation
1)eIF4F is also
regulated many
other ways!
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Post-transcriptional regulation2) Other key step = eIF2
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Post-transcriptional regulation2) Other key step = eIF2• controls assembly of 43S
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Post-transcriptional regulation2) Other key step = eIF2• controls assembly of 43S•Highly regulated!
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Post-transcriptional regulation2) Other key step = eIF2• controls assembly of 43S•Highly regulated!•Inactive if kinased= virus defence
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate• some adopt 2˚ structures hard to melt
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate• some adopt 2˚ structures hard to melt
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate• some adopt 2˚ structures hard to melt• proteins bind others, enhance or repress translation
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate• some adopt 2˚ structures hard to melt• proteins bind others, enhance or repress translation•microRNA bind specific mRNA & block translation
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Post-transcriptional regulationinitiation of translation varies >10x• 5' UTR sequences also affect rate• some adopt 2˚ structures hard to melt• proteins bind others, enhance or repress translation•microRNA bind specific mRNA & block translation•Many bind 3’UTR!
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Post-transcriptional regulation1) mRNA processing2) export from nucleus3) mRNA degradation 4) mRNA localization5) initiation of translation varies >10x6) regulating enzyme activity• activators•Inhibitors•Covalent mods
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Protein degradationSome have motifs marking them for polyubiquitination:• E1 enzymes activate ubiquitin• E2 enzymes conjugate ubiquitin• E3 ub ligases determine specificity, eg for N-terminus
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Protein degradation• E3 ub ligases determine specificity• >1300 E3 ligases in Arabidopsis• 4 main classes according to cullin scaffolding protein
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DWD ProteinsJae-Hoon Lee’s research• putative substrate receptors for CUL4-based E3 ligases
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DWD ProteinsJae-Hoon Lee’s research• putative substrate receptors for CUL4-based E3 ligases• used bioinformatics to find all Arabidopsis & rice DWDs
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DWD Proteins• used bioinformatics to find all Arabidopsis & rice DWDs•Placed in subgroups based on DWD sequence
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DWD Proteins• used bioinformatics to find all Arabidopsis & rice DWDs•Placed in subgroups based on DWD sequence•Tested members of eachsubgroup for DDB1 binding
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins•Only get transcription ifone hybrid supplies Act D& other supplies DNABinding Domain
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DWD ProteinsTwo-hybrid libraries are used to screen for protein-protein interactions
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Cul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadSimilar phenotypes
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DWD ProteinsCul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadsimilar phenotypesPRL1 may be receptor for AKIN10 degradation (involved in sugar sensing)
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DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA
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DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA• ABI5 was elevated in dwa mutants
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DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA• ABI5 was elevated in dwa mutants•ABI5 was degraded more slowly in dwa extracts
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DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA• ABI5 was elevated in dwa mutants•ABI5 was degraded more slowly in dwa extracts•DWA1 & DWA2 target ABI5 for degradation