RNA folding is affected by transcription elongation. As the RNA is synthesized within the elongation complex, its ability to fold is constrained. This can affect downstream processes like intrinsic transcription termination that depend on the formation of RNA hairpin structures. Experiments using RNase T1 to probe RNA structures in elongation complexes at different positions show that the hairpin involved in intrinsic termination begins to form early but may not fully fold until after the RNA is released from the elongation complex. Competing RNA-RNA interactions or kinetic constraints of hairpin folding within the elongation complex can influence the efficiency of intrinsic termination.

1. RNA Folding during Transcription and Its Effect on Intrinsic Termination Natalia Komissarova January 11, 2011

2. Natalia Komissarova Lucyna Lubkowska Anu Mahardjan Mikhail Kashlev Robert Weisberg NCI, Frederick, MD NICHD, Bethesda, MD

3. RNA conformation is crucial for its functionality - translation - plasmid replication - RNA splicing - RNAi-induced gene silencing - gene control by riboswitches - transcription pausing - transcription antitermination - transcription termination

4. The rate of RNA elongation affects the folding pathway The rate of RNA elongation affects the folding pathway because the 5’ end of RNA can fold before the 3’ end is synthesized . Accumulation of metastable folding intermediates can create a kinetic trap

5. Functional consequences of a kinetic trap in RNA folding alternative splicing inactive RNA conformations regulation of time sensitive biological processes like translation and ribosome assembly . Time sensitive processes that occur co-transcriptionally, such as RNA splicing, transcription pausing, termination, and antitermination

6. RNA folding occurs in the elongation complex downstream DNA duplex transcription RNA upstream DNA duplex transcription bubble RNA-DNA hybrid -8 nt -14 nt 5’ 3’ RNAP active center -15 nt +20 nt

7. The termination hairpin UUUUUUUU RNA hairpin oligo U track Intrinsic bacterial terminator h+7 h+8

8. ß flap ß’ rudder downstream DNA duplex template strand nontemplate strand transcription Korzheva et al., Science, 2000 secondary channel RNA exit channel -14 RNA folding occurs in the elongation complex

9. ß flap ß’ rudder transcription RNA -14 Korzheva et al., Science, 2000 RNA folding occurs in the elongation complex

10. Questions How does the elongation complex affect the termination hairpin folding? What is the pathway on the termination hairpin folding? What are the consequences of the constraint on the folding imposed by the elongation complex ?

11. RNase T1 as a tool to assess hairpin folding AATAGCGA 5’NpNpNp Gp NpN 5’NpNpNp Gp + 5’NpN RNaseT1 G1

12. RNase T1 as a tool to assess the hairpin folding G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC G1/mis ATC G CCCTCCTAATAATCGGAGG AA AATAGCGATCATCGCAGCGTACCGAGCGC

13. G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC G1 cleavage in ECs halted downstream of the hairpin

14. G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC AA A A 5’ 5’ 5’ 3’ 3’ 3’ h+6 G1 cleavage in ECh+2, h+5, h+6

15. Komissarova et all., Molecular Cell 2002 h+7 h+8 Hairpin destabilizes ECh+7 and ECh+8

16. 5’ 3’ A Ni-NTA agarose bead - 4nt - 2nt ECh+7 dissociated ECh+7 intact G1 cleavage in ECh+7 5’ 3’ A

17. G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC G1 cleavage in ECh+7 h+7 full size -2 -4

18. G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC G1 cleavage in ECh+8, h+9 h+8 h+9

19. G1 cleavage in ECh+8 containing mismatched hairpin h+8 G1/mis ATC G CCCTCCTAATAATCGGAGG AA AATAGCGATCATCGCAGCGTACCGAGCGC A

20. G3 ATCC G CTCCTAATAATCGGAGCGGAATAGCGATCATCGCAGCGTACCGAGCGC Templates G3, G5, G7 to probe the entire hairpin G5 ATCCCT G CCTAATAATCGGCAGGGAATAGCGATCATCGCAGCGTACCGAGCGC G7 ATCCCCTC G TAATAATCCGAGGGGAATAGCGATCATCGCAGCGTACCGAGCGC

22. Hairpin folding pathway

24. Hairpin folding pathway

26. G1 ATC G CCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC G1 cleavage in ECh+7 h+7 full size -2 -4

27. Hairpin folding pathway

29. Putative RNA structure in ECh+6 based on templates G1, G3, G5, G7 Template G3 Hairpin in ECh+6 Computer-assisted RNA folding G3 ATCC G CTCCTAATAATCGGAGCGGAATAGC h0 h+6 h-7

30. Template G3 Computer-assisted RNA folding Template G3S Hairpin in ECh+6 G3 ATCC G CTCCTAATAATCGGAGCGGAATAGC h0 h+6 h-7 G3 ATCC G C A CCTAATAATCGG U GCGGAATAGC h0 h+6 h-7

31. Template G3S Hairpin in ECh+6 G3 ATCC G C A CCTAATAATCGG U GCGGAATAGC h0 h+6 h-7

32. Hairpin folding pathway Pause EC rearrangement Mismatched hairpin ECh+8

33. Alternative structure affects termination

34. Alternative structure affects termination Template G1L AA

35. Alternative structure affects termination

36. Sequence context affects termination

37. Competing RNA-RNA interactions affect termination

38. RNA-RNA interactions competing with hairpin formation Weak competing RNA-RNA interactions affect termination termination h+7 h+8 h0 h0 Full terminated RNA 3’ GCCUUUUUAU RNA in the EC at termination point tR2/T/long U AUCGAGA CAC CG GGGA GG A GGU CCCU CC A AU CGUCCGG CUAA AC GCAGGCC AUC A UUUUUAU 5’ 3’ h0

39. RNA-RNA interactions competing with hairpin formation Weak competing RNA-RNA interactions affect termination termination h+7 h+8 h0 h0 3’ GCCUUUUUAU RNA in the EC at termination point tR2/T/long

40. Weak competing RNA-RNA interactions affect termination

41. Weak competing RNA-RNA interactions affect termination because of its kinetic nature and the EC interference with hairpin folding Jack Sparrow: If you were waiting for the opportune moment, that was it.

42. - why some mutations that do not alter or increase hairpin strength decrease termination (Cheng et al., Science (1991), 254; Wilson and von Hippel, PNAS (1995), 92) - why the efficiency of terminators depends on early transcribed promoter-proximal sequences (Goliger et al. (1989) J. Mol. Biol. 205; Telesnitsky and Chamberlin (1989) J. Mol. Biol. 205) Models of termination do not explain: