1. MOLECULAR BASIS OF RNA- DEPENDANT RNA POLYMERASE II ACTIVITY Elisabeth Lehmann et. al.

2. Introduction  Catalyses DNA- Dependant RNA synthesis  Structure has 12 subunits ~ 550 kDa  Key polymerase in transcription

3. Introduction  Error Rate : 10 -3 “clamp” swings over DNA to trap it, ensures high processivity Unwound template strand make 90° turn after active site due to “wall” Active site accessible through funnel for new NTPs backbone DNA-RNA hybrid helix is disrupted by “rudder”

4.  B-DNA form entering downstream  A-RNA form exiting upstream

5. RNA Polymerase II and RdRP  Suggested that TFIIS and Pol II have RdRP capabilities  Evolutionary Link!  Look at replication of HDV

6. RdRP Scaffold  The downstream cleft can accommodate A-RNA, as observed for the 3’ stem of the RNA inhibitor FC*  The FC* 3’ stem overlaps with two DNA template positions downstream of the NTP-binding site (position +1) suggesting that an RNA template could enter the active site in a similar manner to DNA

7. RdRP Test!! (YAY!!!)  Prepared RNA scaffold that combined FC* 3’ Stem, with RNA template  Product Strand was labeled at the 5’ End with FAM (6-carboxyfluorescein)  Scaffold + Pol II, NTPs => RNA Elongation!

8. Elongation Results  Elongation by ~8 nt  Misincorporation at +3 and +1  RNA Issue?  DNA produced the same errors  Issue arose due to non cognate NTP

9. Elongation Results  Lane 1. Reactant RNA  Lane 2 – 10 Incubation with different types of NTP  Lane 2-5 RNA scaffold RdRP  Lane 6-9 DNA scaffold DdRP

10. Polymerase II crystal Structure!  Scaffold with 5’ six nt extension suffice to form an active RdRP EC  RNA 3’ was bound to catalytic metal ion A  Structure of RNA Duplex is essentially identical to that of the DNA-RNA Hybrid.

11. RNA and DNA comparison  Time Course Experiment:  RdRP activity much slower  RNA template replication stopped prematurely.  Changing Downstream duplex length was changed, the product length changed accordingly.  Uracil at +9 position was found to cause an interruption

12. Investigation of RdRP activity  Studied Terminal Segment of HDV antigenome  Directs Synthesis and is sensitive to Pol II inhibitor α - amanitin  RNA cleavage followed by elongation of the new 3’ end.  Cleaved segments forms a template-product stem loop

13. HDV Elongation Pol II and NTPs resulted in RNA synthesis up to the end of the template Only cognate NMPs were incorporated at positions11 and12 When UTP was omitted from the reaction, RNA synthesis stopped at position +13 as expected

14. RdRP activity continued  Exclusion of UTP lead to synthesis of RNA up to position +13 as expected  Slow reaction rate  Different reaction rates between the HDV and RdRP scaffold may possibly originate from different upstream template product duplex.

15. Here comes the chimaeric Scaffolds!  Recombine upstream and downstream regions of both scaffolds  RdRP upstram + HDV downstream = low processivity  RdRP downstream + HDV upstream = Run off synthesis that was not possible with RdRP scaffold.  Stem loop + 2nt 5’ extension forms a functional EC

16. RdRP and HDV processivity HDV upstream and RdRP downstream HDV downstream and RdRP upstream

17. RdRP- Promoting Stem Loop  To test whether the RdRP-promoting stem-loop can be formed from the HDV terminal segment  Incubate HDV terminal segment with PolII and TFIIS  Check for RdRP promoting Stem – Loop  Resulted in RNA cleavage at the internal buldge, which connected the RNA duplexes in a flexible way.  This allows for positioning of the RNA at the active site  Product is a 6 bp stem loop comparing with the 5bp stem loop that forms in extracts  Perfect RdRP Activity!

18.  HDV-derived terminal stem-loops consisting of 5 or 6 bp enable templated incorporation of the next nucleotide

19. RdRP processivity  “Consistently, Pol II readily used stem-loops with 5, 6, 7 or 10 bp as substrates, but not stem-loops with 13, 15 or 18 bp”  “The limited RdRP processivity in vitro is apparently overcome during HDV replication in vivo by binding of the HDV encoded elongation-stimulatory delta antigen to the polymerase Clamp”

20. Stem Loops and Processivity Checking for RdRP activity at different stem loop lengths

21. Conclusion  Less processive that DdRP  Pol II cleaves 1 HDV strand and creates a reactive stem loop in the hybrid site, and extend the RNA 3’ site  During Transcription, upstream template and product strands are separated, whereas the HDV stem loop probably persists during elongation.  Pol II readily uses stem loops with 5-10bp, but nothing of larger length.  A persistent RNA-DNA hybrid stalls transcription EC  RdRP stalls in vitro is overcome during HDV replication in vivo by binding of the HDV encoded elongation-stimulatory delta antigen to the polymerase clamp.

22. Discussion  RdRP processivity of RNA II was discovered  However products have to be less than ~10bp  Vitro results in HDV catalytic stem loop length are different than that of vivo  Evolutionary link is established

23. Further Research  Segregate the enzyme responsible for allowing full RdRP activity (believed to be antigen delta).  Determine the crystal structure of the RdRP cofactor!!  Evolutionary link established???