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Post-transcriptional Control of Chloroplast Gene Expression via RNA-Protein Complexes Current Josh Baecker Patricia Merhige Brandon Stilb Department of Energy Past Dawn Both Tracy Hotchkiss Mark Robida Chloroplasts are like big green prokaryotes Spinach Chloroplast genome: 150,725 bp However, the majority of chloroplast polypeptides are encoded by the nucleus. Post-transcriptional Mechanisms Have Important Effects on Chloroplast Gene Expression RNA processing RNA stability Translation Mediated by RNA-protein Complexes Protein modification/complex assembly Protein turnover RNA-protein Complexes Can Form in 5’Untranslated Regions (5’UTRs) 5’UTRs are sequences upstream of open reading frames In chloroplasts, they often encode ribosome binding sites. Proteins that bind to chloroplast 5’UTRs have been shown to affect RNA stability and/or translation initiation. RNA-protein Complexes Can Be Analyzed by Gel Mobility Shift Assays + RNAs Potential binding proteins (specific, radiolabeled) (non-specific) (specific) (non-specific) (non-binding) Incubation Self competition: Chloroplast extract: RNA-protein complexes { Native gel Unbound RNA: - + + + Questions to Be Answered About Chloroplast RNA-protein Complexes What are the cis-acting elements that affect complex formation? What are the proteins involved in the complexes? What is the function of these complexes? What are the cis-acting elements that affect complex formation? Is there conserved RNA structure? Where do the proteins bind? The atpI 5’UTR Is Our Baseline Binding Substrate Arabidopsis Calycanthus Lotus Maize Nightshade Pea Primrose Rice Tobacco Wheat Spinach Consensus .......... .........G GAAATTTTTG .......... .......... .......... .........G .........G .......... .........G .......... .......... .......... GTTACTATCC ATTAATAATA .......... CTTACTATTT ..GGGTATTA GTTACTACTT GGGAATATTG .......... GGGAATATTG ......GATT .......... .......... CTGAATCTTA AAATAAAAAA .......... CTGAATTTCA TAGCATTACA TTGAATCGCA ATATATATTA .......... ATATATATTA TTGAATCTCA ....a....a .......... .AAAAGAGAT ATAAAGAGAA ....GGGTAT AAAAAGAGAT AAATTGTGTA TAAAAGAGAT ..GAGGGTAT .......... ..GAGGGTAT AAAACTAGTT .......... .....GGGGA AAAAATAAGT TAAAGAACAA TGATATATAT AAAAATAACT AAAAGAA... CAAAATGGAT TGATATATAT .......... TGATATATAT CAAAATAACA ......R... Arabidopsis Calycanthus Lotus Maize Nightshade Pea Primrose Rice Tobacco Wheat Spinach Consensus TATTGTGTGA GGGGATATTA ACAGAAATAT TATGATCTGA GGGGATATTA ....ATATTT GGAGATGCTG TATCATCTGA GGGGATATTA TATGATCTGA GGGGATATTA ....AT.T.. TTTGTTTAGT TGCGATCGCT TATG..TGAT TGTGATTTCT GGTGATTTAT TGTGATTAGT TGTGATTGGT TGTGATTTCT TGTGATTTAT TGTGCTTTCT TGTAATTAAT TgTG.TT..T TGGGATCCAA TGGTATCCGA TAGTAGATAA TGGTATATTA TAGTATTCGA AGGTATTCAA TAGTATACAA TGATATCCTA TAGTATTCTA TGGTATCCTA CAGTATCCGA tRGTAT.c.A AAC.TAAAAT AATACACGAT AATAGAAAAT AATATAAGAT AATCTTAG.T AATAGAAAAT AATAGAAAAT AATATAAGAT AATCTTAG.T AATATAAGAT AATAGAAAAT AAT..aARaT ATAAAATTTA TAA.AGTAGG CAA.AGTAGA TAATACTTCA TGGTATTCAA CAA....... TCA.ACTAAG TAATACTTCA TGGTATTCAA TAATACTTCA TTT.AATTAA Y.R.a.T..R Arabidopsis Calycanthus Lotus Maize Nightshade Pea Primrose Rice Tobacco Wheat Spinach Consensus AGTAAATAAG GG....CGAG ......CAAG AGTTGCTGAG AATATCCGAT ........AG ......CAAG CGTTGCTGAG AATATCCGAT AGTTGCTGAG AGTAGACAAG ......YRAg TAAAAAAAAA CCGAGAA... TCAAAAA... TTGAGAA... TCAAGTAGAC TAAAATA... TCAAAAA... TTGAGAA... TCAAGTAGAC TTGAGAA... TCGAGAA... TyRAR.A... GGGGGGGT.C .AG.AGATGT .AGGAGATGA .AG.AGATGC AAAGAGATGG .AGGAAATGG .AG.AGATGG .AA.AGATGG AAAGAGATGG .AG.AGATGG .AG.AGCTGA .AR.AGaTG. TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA TTGAATCAAA ATAATTTA.. ATAATTCCTT ATAATTCCCT AGAATTCCTT AAATTTTGTT ATAATTCCCT ATAATTCCTT AGAATTCTTT AAATTTTGTT AGAATTCCTT ATAATT..TT A.AaTTy.yT Arabidopsis Calycanthus Lotus Maize Nightshade Pea Primrose Rice Tobacco Wheat Spinach Consensus ........AA TTT.....AA TTC.....AA TTT...TGAA TA......AA TTCAAGTTAT TTA.....AA TTT...TGAA TA......AA TTT...TGAA TTT.....AA T.......AA GTTCTTATTT GTTCT.ATTT GTTCT.ATTT GTTCA.ATTT GTTCA.ATTT ATT.T.TTTT GTTCG.ATTT GTTCA.TTTT GTTCA.ATTT GTTCA.ATTT GTTAT.ATTT GTTc..aTTT CTGTCAGAGG CTGTCAGAGG CGTTTAGAGG TTATCAGAGG TT.TCAGAGG ATTTTAGAGG ATGTGAGAGG TTATCAGGGG TT.TCAGAGG TTATCAGAGG CTGTAAGAGG yT.T.AGAGG GC.....AAT GC.....AAT GCGTGGCAAT AC.....AAT GCAAGGCAAT ACAGGGCAAT GC.....A.T AC.....AAT GCAAGGCAAT AC.....AAT AC.....AAT RC.....AAT ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG THERE ARE TWO BINDING SITES IN THE atpI 5’UTR ORF -155 -52 -1 +15 C: + - E: + + * * ORF -155 C: + - E: + + - -58 -52 C: + - E: + + - * * C: self-competitor E: chloroplast extract *: RNA-protein complex -1 +15 What are the proteins involved in the complexes? Are they ATP synthase-specific? Competition Binding Assays Revealed that the Same Chloroplast Proteins Bind Diverse 5’UTRs Good Competitors; similar binding proteins atpI ATP synthase subunit CFo-IV atpA ATP synthase subunit CF1-α atpE ATP synthase subunit CF1-ε Partial competitor; some shared proteins atpF ATP synthase subunit CFo- I atpH ATP synthase subunit CFo-III rps16 small ribosome subunit 16 clpP protease subunit infA translation initiation factor Poor competitor; few shared proteins ndhD NADH dehydrogenase subunit psbA Photosystem II D1 protein petL cytochrome b/f 3.5 kDa subunit psaC photosystem I 9 kDa protein rbcL large subunit, carboxylase rpl22 large ribosome subunit protein 22 Supershift rpoA RNA polymerase α-subunit atpB ATP synthase subunit CF1-β rpoB RNA polymerase β-subunit rps2 small ribosome subunit protein 2 rps11 small ribosomal subunit 11 Affinity purification of binding proteins atpI 5’UTR Streptavidinbinding motif Chloroplast extract Incubate S Isolate mag bead SDS-PAGE of affinity-isolated 5’UTR-binding proteins MW Unbound W1 66 55 42 37 27 20 14 W2 W3 Isolated What is the function of these complexes? Do they affect translation? Do they affect RNA stability? Chloroplast Transformation Target Plants (Week 2) Second Selection (Week 10) Antibiotic-resistant transformants (Week 6) Rooting (Week 12) Roots Variations on a single 5’UTR Wild-type D Deletion Chimeric Complete deletion (except for RBS) D misc. 5’ UTR atpI sequence deletion 5’ UTR uidA reporter rps16 3’ UTR The atpI 5’UTR Affects Translation and RNA Abundance Conclusions & Projects Cis-acting elements There are two binding sites and extensive phylogentically conserved sequence in the atpI 5’UTR Structure probing studies will be used to identify specific structure(s) necessary for binding. Trans-acting factors The same polypeptides bind many chloroplast 5’UTRs. Affinity-based methods will be used to isolate the binding protein(s) Once the genes encoding them are cloned, they will be mutated and expressed in vivo Function Preliminary data demonstrate an effect of the atpI 5’UTR on both RNA stability and translation. In vivo experiments will be used to dissect 5’UTR function.