RNA Folding

RNA Folding during Transcription

and Its Effect on Intrinsic Termination

Natalia Komissarova

January 11, 2011

Natalia Komissarova

Lucyna Lubkowska

Anu Mahardjan

Mikhail Kashlev

Robert Weisberg

NCI, Frederick, MD

NICHD, Bethesda, MD

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

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

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

RNA folding occurs in the elongation complex

downstream DNA duplex

transcription

RNA

upstream DNA duplex

transcription bubble

RNA-DNA hybrid

-8 nt-14 nt5’

3’

RNAP active center

-15 nt

+20 nt

The termination hairpin

UUUUUUUU

RNA hairpin

oligo U track

Intrinsic bacterial terminator

h+7 h+8

ß flap

ß’ rudder

downstream DNA duplextemplate strandnontemplate strand

transcription

Korzheva et al., Science, 2000

secondary channel

RNA exit channel

-14


ß flap

ß’ rudder

transcription

RNA

-14

Korzheva et al., Science, 2000


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 ?

RNase T1 as a tool to assess hairpin folding

AATAGCGA

5’NpNpNpGpNpN 5’NpNpNpGp + 5’NpNRNaseT1

G1

G1ATCGCCCTCCTAATAATCGGAGGGCAATAGCGATCATCGCAGCGTACCGAGCGC1

Time withRNase T1, min

- 1 3 10 20 - 1 3 10 20 G1G1/misTemplate1 2 3 4 5 6 7 8 9 10 Free RNA isolated from EC h+5h+5 full size30 nt

h+2 full size30 nt

h+2 cut at G126 nt

G1G1h0A5’3’ G1G1/mish05’A3’

G1/misATCGCCCTCCTAATAATCGGAGGAAAATAGCGATCATCGCAGCGTACCGAGCGC 1

RNase T1 as a tool to assess the hairpin folding

- 1 3 10 20 20h+2Time with RNaseT1, min

ECTemplate G1- 1 3 10 20 20- 1 3 10 20 20- 1 3 10 20 20- 1 3 10 20 - 1 3 10 20 20- 1 3 10 20 20h+5h+6h+7h+7h+8h+9wwwwwwfree RNA1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41* h+2 full sizeh+2 cut at G1


h0h+2h+5

h+7h+9

G1 cleavage in ECs halted downstream of the hairpin




h+2h+5

AGAGAAh0ECh+2G1GG GGGECh+6G1ECh+5G1GG GGGGG GGG

AA

A

A

5’

5’

5’

3’

3’

3’

h+6

G1 cleavage in ECh+2, h+5, h+6

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Komissarova et all., Molecular Cell 2002

h+7 h+8

Hairpin destabilizes ECh+7 and ECh+8

5’ 3’

AG1Gh+5GGGGG

A

Ni-NTA agarose bead

- 4nt

- 2nt

5’

3’

AGECh+7G1GGGGG

A

ECh+7 dissociated

ECh+7 intact

G1 cleavage in ECh+7




h+7


h+7 full size-2-4




G1 cleavage in ECh+8, h+9

h+8 h+9

h+5h+7h+8- 1 3 20 40 - 1 3 20 40 - 1 3 20 40 Time with RNaseT1, min

EC1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 h+5 full sizeh+5 cut at G1Template G1/mis

G1/misATCGCCCTCCTAATAATCGGAGGAAAATAGCGATCATCGCAGCGTACCGAGCGC 1

G1 cleavage in ECh+8 containing mismatched hairpin

G1/mis

G1h05’A3’

A

h+8

G3 ATCCGCTCCTAATAATCGGAGCGGAATAGCGATCATCGCAGCGTACCGAGCGC

3

G5 ATCCCTGCCTAATAATCGGCAGGGAATAGCGATCATCGCAGCGTACCGAGCGC

5

G7 ATCCCCTCGTAATAATCCGAGGGGAATAGCGATCATCGCAGCGTACCGAGCGC

7

Templates G3, G5, G7 to probe the entire hairpin

h+2h+5

h+7h+9

EC- 1 3 10 20 20h+2Template G3- 1 3 10 20 20- 1 3 10 20 - 1 3 10 20 20- 1 3 10 20 20 - 1 3 10 20 20h+5h+6h+7h+8h+9wwwwww Time with RNaseT1, min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 * h+2 full sizeh+2 cut at G3

h+5h+6h+7h+8- 1 3 10 20 20w- 1 3 10 20 20w- 1 3 10 20 20w- 3 10 20 20w EC Time with RNaseT1, min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Template G5* * h+5 full sizeh+5 cut at G5

EC Time with RNaseT1, min

Template G7- 1 3 10 20 20h+2- 1 3 10 20 20- 1 3 10 20 20 - 1 3 10 10- 1 3 10 20 20 - 1 3 10 20 20h+5h+6h+7h+8h+9wwwwww1 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36* h+2 full sizeh+2 cut at G7

17531753ECh+5ECh+6h01735ECh+77351ECh+8

Hairpin folding pathway







17531753ECh+5ECh+6h01735ECh+77351ECh+8











h+7


h+7 full size-2-4

h+7 full sizeh+7 cut at G1Time with

RNaseT1, minEC

Template G1

17531753ECh+5ECh+6h01735ECh+77351ECh+8








h+6G1G3G5G7h0

Putative RNA structure in ECh+6 based on templates G1, G3, G5, G7

dG=-1.1 kcal/mol U AA AA U U C C G C G U A C G3 C C

AUGCGG

AAUAGCh-4dG=-1.8 kcal/mol U A A C U C A U A C U G3C C G C G AU A

GCGGAAUAGCh-4

Template G3

G3 ATCCGCTCCTAATAATCGGAGCGGAATAGC

3

h0 h+6h-7

Hairpin in ECh+6

Computer-assisted RNA folding

dG=-0.3 kcal/mol A:U inversion in the stem U A A C U C A A A C U G3C C GAUC GU

GCGGAAUAGCAUGCGG

AAUAGCdG=-1.1 kcal/mol U AA AA U U C C G C G A U C G3 C C

extremely unstable dG=-1.1 kcal/mol U AA AA U U C C G C G U A C G3 C C

AUGCGG

AAUAGCh-4dG=-1.8 kcal/mol U A A C U C A U A C U G3C C G C G AU A

GCGGAAUAGCh-4

Template G3

G3 ATCCGCTCCTAATAATCGGAGCGGAATAGC

3

h0 h+6h-7

Computer-assisted RNA folding

Template G3S

G3 ATCCGCACCTAATAATCGGUGCGGAATAGC

3

h0 h+6h-7

Hairpin in ECh+6

dG=-0.3 kcal/mol A:U inversion in the stem U A A C U C A A A C U G3C C GAUC GU

GCGGAAUAGCAUGCGG

AAUAGCdG=-1.1 kcal/mol U AA AA U U C C G C G A U C G3 C C

extremely unstable

Template G3S

G3 ATCCGCACCTAATAATCGGUGCGGAATAGC

3

h0 h+6h-7

Figure 3Time with RNaseT1, min

ECh+6h+7 and h+6h+8 and h+6- 1 3 10 20 20 W

- 1 3 10 20 20 W

- 1 3 10 20 20 W

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 * h+6 full sizeh+6 cut at G3

Hairpin in ECh+6

17531753ECh+5ECh+6h01735ECh+77351ECh+8


Pause

EC rearrangement

Mismatched hairpin

7351

ECh+8

G1/T ATCGCCCTCCTGGTAATCGGAGGGCTTTTTATTTCATCGCAGCGTACCGAGCGC G3/T ATCCGCTCCTGGTAATCGGAGCGGTTTTTATTTCATCGCAGCGTACCGAGCGC

h+8terminationh+7h0

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture. G1

/T G3/TKCl4 NTPs, mMTemplate 300 mM 0.1 0 .01 0.1 0.01Runoff1 2 3 4 Terminationh+8h+7

Alternative structure affects termination

Time withRNaseT1, minh+2- 1 3 10 20 20h+8, h+9ww- 1 3 10 20 20 EC1 2 3 4 5 6 7 8 9 10 11 12 h+9 fullh+8 fullh+8 cut at G1

A A U U C C G C G U A C G C G

CCAAUAGCGAAUCGCCCU U AC AC U U C C G C G U A C G C G C G G C

AUCGCAAUAGCGA11dG=-13.9 kcal/moldG=-7.0 kcal/mol


Template G1L

AA


Template G1/T G1L/Ts ps pRunoffFraction300 mM KCl, 1 mM NTPs

1 2 3 4Terminationh+8h+7

G1/T ATCGCCCTCCTGGTAATCGGAGGGCTTTTTATTTCATCGCAGCGTACCGAGCGC h+8terminationh+7h0

G1L/TATCGCCCTCCTCCTAATCGGAGGGCTTTTTATTTCATCGCAGCGTACCGAGCGC

5’ATCGAGAGGGACACGGCGAATACCCATCCCAATCGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGATCGCAGCGTACCGAGCGC3’tR2/T/longh+7h+8h0tR2/T 5’ ATCGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGATCGCAGCGTACCGAGCGC3’ terminationh+7h+8h0termination

Sequence context affects termination

0102030405060708090100

0 3 10 30100

3’ATCGAGAGGGACACGGCGAATACCCATCCC5’ - control oligo3’TAGCTCTCCCTGTGCCGCTTATGGGTAGGG5’ - complementary oligo 0 3 10 30 100Oligo concentration, μM 2/ / (0.1 , 300 )Termination on tR T long template mM NTPs mM KCl5’ ATCGAGAGGGACACGGCGAATACCCATCCCAATCGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGATCGCAGCGTACCGAGCGC3’2/ /tR T long+7h+8h0htermination

Competing RNA-RNA interactions affect termination

RNA-RNA interactions competing with hairpin formation

Weak competing RNA-RNA interactions affect termination

termination

5’ATCGAGAGGGACACGGCGAATACCCATCCCAATCGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGATCGCAGCGTACCGAGCGC3’

h+7 h+8h0

UU

AUCGAGA CAC AUCGAGA CAC

CG CG

GGGA GGGA

GG AGG A GGUGGU

CCCU CCCU

CC ACC A

AUAU CGUCCGGCGUCCGG

CUAA AC CUAA AC

GCAGGCCGCAGGCC AUCAUC

AA UUUUUAUUUUUUAU

5’

3’

h0

A GA CAC CG UCGA GGGA GG A GGCU CCCU CC A AA AC AU

5’3’GCCUUUUUAUh0 GGU CU CGUCA GCAG AUC

h0

Full terminated RNA

3’ GCCUUUUUAUGCCUUUUUAU

RNA in the EC at termination point

tR2/T/long

RNA-RNA interactions competing with hairpin formation


termination

5’ATCGAGAGGGACACGGCGAATACCCATCCCAATCGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGATCGCAGCGTACCGAGCGC3’

h+7 h+8h0

A GA CAC CG UCGA GGGA GG A GGCU CCCU CC A AA AC AU

5’3’GCCUUUUUAUh0 GGU CU CGUCA GCAG AUC

h0

3’ GCCUUUUUAUGCCUUUUUAU

RNA in the EC at termination point

tR2/T/long

3’AUCGAGAGGGACACGGCGRNase ARNase T1ECh-365 ’

Template tR2/T/longEC[h-36]RunoffTermination4 NTPs, mM 0.1 1 0.1 1 0.1 112345620 nt17 nt14 nt


TerminationReadthrough1735ECh+77351ECh+8

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.

- 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:

Technology

RNA Folding