prepare about a 10 minute talk for wednesday feb 25 on something to do with ncrna, rna editing,...
TRANSCRIPT
Prepare about a 10 minute talk for Wednesday Feb 25 on something to do with ncRNA, RNA editing, alternative
poly-adenylation, mRNA localization or mRNA transport.
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
Post-transcriptional regulationProtein degradation rate varies 100x• Some have motifs, eg Destruction box, marking them for
polyubiquitination: taken to proteasome & destroyed
DWD ProteinsJae-Hoon Lee’s research• putative substrate receptors for CUL4-based E3 ligases
DWD Proteins• used bioinformatics to find all Arabidopsis & rice DWDs•Placed in subgroups based on DWD sequence•Tested members of eachsubgroup for DDB1 binding
DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation
DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins
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
DWD ProteinsTwo-hybrid libraries are used to screen for protein-protein interactions
DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid
DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Cul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadSimilar phenotypes
DWD ProteinsCul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadsimilar phenotypesPRL1 may be receptor for AKIN10 degradation (involved in sugar sensing)
DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA
DWD Proteins•Found T-DNA insertions
•3 were sensitive to ABA• ABI5 was elevated in dwa mutants
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
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
Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullin
Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullin
Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8
Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8Regulates DNA-damage response, cell-cycle & gene expression
Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8Regulates DNA-damage response, cell-cycle & gene expressionNot all E3 ligases associate withCullins!
COP1 is a non-cullin-associated E3 ligase• Protein degradation is important for light regulation• COP1/SPA1 tags transcription factors for degradation• W/O COP1 they act in dark• In light COP1 is exported to cytoplasm so TF can act
COP1 is a non-cullin-associated E3 ligase• Recent data indicates that COP1 may also associate
with CUL4
Protein degradation rate varies 100xMost have motifs marking them for polyubiquitination:
taken to proteosome & destroyedOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain
Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x
Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub
Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role
Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role
• KFERQ: cytosolic proteins with KFERQ are selectively taken up by lysosomes in chaperone-mediated autophagy under conditions of nutritional or oxidative stress.
Protein degradation in bacteriaAlso highly regulated, involves chaperone-like proteins1.Lon
Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp
Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp3.FtsH in IM
PROTEIN TARGETINGAll proteins are made with an “address” which determines their final cellular location
Addresses are motifs within proteins
PROTEIN TARGETINGAll proteins are made with “addresses” which determine their locationAddresses are motifs within proteins
Remain in cytoplasm unless contain information sending it elsewhere
PROTEIN TARGETING Targeting sequences are both necessary & sufficient to send reporter proteins to new compartments.
PROTEIN TARGETING2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!
2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK
2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol
2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol•Binds Tat in IM & is sent to periplasm
2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol•Binds Tat in IM & is sent to periplasm•Signal seq is removed inperiplasm
2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!2.Sec pathway•SecB binds preproteinas it emerges from rib
Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding
Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding•Guides it to SecA, which drives it through SecYEG into periplasm using ATP
Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding•Guides it to SecA, which drives it through SecYEG into periplasm using ATP•In periplasm signal peptide is removed and protein folds
Sec pathway part deux•SRP binds preprotein as it emerges from rib & stops translation•Guides rib to FtsY•FtsY & SecA guide it to SecYEG , where it resumes translation & inserts protein into membrane as it is made
Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system