1. Post-transcriptional Control of Chloroplast Gene Expression via RNA-Protein Complexes CurrentPast Josh BaeckerDawn Both Patricia MerhigeTracy Hotchkiss Brandon StilbMark Robida Department of Energy

2. Chloroplasts are like big green prokaryotes However, the majority of chloroplast polypeptides are encoded by the nucleus. Spinach Chloroplast genome: 150,725 bp

3. Post-transcriptional Mechanisms Have Important Effects on Chloroplast Gene Expression RNA processing RNA stability Translation Protein modification/complex assembly Protein turnover Mediated by RNA-protein Complexes

4. 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.

5. RNA-protein Complexes Can Be Analyzed by Gel Mobility Shift Assays RNAs Potential binding proteins (specific, radiolabeled) (specific) (non-specific) (non-specific) (non-binding) Incubation Native gel Self competition: - - + Chloroplast extract: - + + Unbound RNA: { RNA-protein complexes +

6. 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? Questions to Be Answered About Chloroplast RNA-protein Complexes

7. What are the cis-acting elements that affect complex formation? Is there conserved RNA structure? Where do the proteins bind?

8. The atpI 5’UTR Is Our Baseline Binding Substrate

9. C: + - - E: + + - * * C: + - - E: + + - * THERE ARE TWO BINDING SITES IN THE atpI 5’UTR ORF -155 -52 -1 +15 -155 -58 C: + - - E: + + - * -52 -1 +15 ORF C: self-competitor E: chloroplast extract *: RNA-protein complex

10. What are the proteins involved in the complexes? Are they ATP synthase-specific?

11. Competition Binding Assays Revealed that the Same Chloroplast Proteins Bind Diverse 5’UTRs Good Competitors; similar binding proteins atpIATP synthase subunit CF o -IV atpAATP synthase subunit CF 1 -α atpEATP synthase subunit CF 1 -ε atpFATP synthase subunit CF o - I atpHATP synthase subunit CF o -III clpPprotease subunit infAtranslation initiation factor ndhDNADH dehydrogenase subunit petLcytochrome b/f 3.5 kDa subunit psaCphotosystem I 9 kDa protein rbcLlarge subunit, carboxylase rpl22large ribosome subunit protein 22 rpoARNA polymerase α-subunit rpoBRNA polymerase β-subunit rps2small ribosome subunit protein 2 rps11small ribosomal subunit 11 Poor competitor; few shared proteins psbAPhotosystem II D1 protein Partial competitor; some shared proteins rps16small ribosome subunit 16 Supershift atpBATP synthase subunit CF 1 -β

12. Affinity purification of binding proteins Streptavidin- binding motif atpI 5’UTR Chloroplast extract S mag bead Incubate Isolate

13. MW Unbound W1 W2 W3 Isolated 66 55 42 37 27 14 20 SDS-PAGE of affinity-isolated 5’UTR-binding proteins

14. What is the function of these complexes? Do they affect translation? Do they affect RNA stability?

15. Chloroplast Transformation Target Plants (Week 2) Antibiotic-resistant transformants (Week 6) Second Selection (Week 10)Rooting (Week 12) Roots

16. uidA reporter rps16 3’ UTR  5’ UTR deletion atpI 5’ UTR misc. sequence Chimeric Wild-type Deletion  Variations on a single 5’UTR Complete deletion (except for RBS)

17. The atpI 5’UTR Affects Translation and RNA Abundance

18. 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.