1. RNA-catalysed nucleotide synthesis Peter J. Unrau & David P. Bartel Pamela Lussier Biochemistry 4000/5000

2. ‘RNA World’ Hypothesis  Hypothetical stage in origin of life on Earth.  Proposes that early life developed by making use of RNA molecules to store information (DNA) and catalyze reactions (proteins)  Thought that nucleotides constituting RNA were scarce on early Earth

3. RNA-based life synthesized RNA from precursors

4. RNA nucleotide synthesis  Prebiotic synthesis routes previously proposed for sugars, sugar phosphates, and the four RNA bases.  Still a Challenge – coupling the molecules into nucleotides.

5. Modern Metabolism Activated Ribose Pyrimidine Base Pyrimidine Nucleotide

6.  Release of pyrophosphate from activated ribose causes nucleophilic attack on carbon  Metabolic pathway forms both nucleotides and amino acids tryptophan and histidine in modern metabolism  This mechanism is absent from known ribozyme reactions.

7.  Unique to known RNA-catalysis:  Occurs by S N1 reaction mechanism  Uracil is significantly smaller than the smallest ribozyme substrate.

8. Figure 2 Pre-Adenylylation bypasses the specificity for donor substrate of T4 RNA ligase Thione reacts strongly with thiophilic reagents Denaturing gel, impedes migration of RNA containing 4-thioU Reacts with –SH group to form stable thioether linkage

9. Steps for in vitro selection  pRpp attatched to 3’ end of pool RNA  RNA-pRpp incubated with a 4-thiouracil (uracil analogue)  RNA attached to newly synthesized nucleotide 4-thiouridine were enriched, amplified  Process of selection-amplification again

10. Triangle = uncatalyzed reaction rate After 4 rounds = ribozyme activity readily detected Round 4-6 = error prone PCR amplification Round 7-10 = decreasing the 4S Ura concentration and decreasing the incubation time Ribozyme activity

11. Ribozymes after 11 rounds of selection were cloned 35 random clones were sequenced Family: A – 25 B – 8 C – 2 Restriction analysis of PCR DNA indicated that these were the only three families of nucleotide-synthesizing ribozymes to immerge

12.  To detect uncatalyzed reaction – radio- labelled pRpp-derivatized oligonucleotide was incubated with 4S Ura and reaction mixture was resolved on AMP gel  Result = nothing detected  Gel could detect rates as slow as 6 x 10 ^- 7 M -1 min -1

13. Michaelis-Menten Kinetics  K M = Michaelis constant. Equal to the [S] at which the reaction rate is ½(V max ).  E + S ES P + E k1 K-1 k2

14.  Enzyme’s Kinetic parameters provide a measure of its catalytic efficiency  Kcat = Vmax/[E] T  Number of rxn processes each active site catalyzes per unit time  When [S]<<Km, little ES is formed  [E] ~[E] T so equation below can reduce to a second order rate equation: Vo = k2[ES] = (k2[ET][S])/(KM + [S]) Can become: Vo = (Kcat/Km)[E][S]

15.  Kcat/Km is the second-order rate constant of enzymatic reaction  Varies with how often enzyme and substrate encounter each other  So kcat/Km is measure of enzymes catalytic efficiency

16. Isolates from each family promoted nucleotide formation up to 10 ^7 times greater than upper bound on uncatalysed reaction rate.

17. Circle = Family A – a15 Square = Family B – b01 Diamond = Family C – c05 Fits to a Michaelis- Menten curve Do not display saturable behavior Suggests poorer binding to 4S Ura

18.  Above14 mM – cannot measure due to solubility constraints.  Cannot discount possibility that 4S Ura was starting to occupy inhibitory site, rather than catalytic site.  Linear behavior of family b and c suggest 4S Ura doesn’t aggregate of affect metal- ion availability.

19. High Specificity for 4S Ura  Incubated all three ribozymes with thio- substituted bases (2-thiouracil, 2,4- thiouracil, 2-thiocytosine, 2-thiopyrimidine, 2-thiopyridine, and 5-carboxy-2-thiouracil)  No thio-containing product detected on AMP gel.

20. Jump back to Proteins  Thought to catalyze rxn by stabilizing oxocarbocation at the C1- carbon of reaction center  Challenge: avoiding hydrolysis  Can avoid by excluding water from active site, and promoting carbocation formation only after conformational change  What about Ribozymes?

21.  Examine degree of hydrolysis of tethered pRpp  Promoted hydrolysis 12-23 x faster than uncatalysed hydrolysis  Rates for 4S Ura formation were ≥60 times faster than rates of catalysed hydrolysis.

22.  RNA could have new strategy to promote glycosidic bond formation by stabilizing TS with more S N2 character

23. Cofactors?  All three ribozyme families required divalent cations for activity.  Each round Mg 2+, Mn 2+ and Ca 2+ provided.  Ca 2+ dispensable for all families  All preferred Mg 2+ over Mn 2+

24.  Family A did not need Mn 2+ (twofold decrease in activity in absence of)  Family B and C require Mn 2+, with the presence of 25mM Mg 2+ reaching a plateau at 1mM Mn 2+  Family B ribozyme did not require for stimulating pRpp hydrolysis – Mn 2+ has a role in binding or proper orientation of the 4S Ura consistent with the thiophilic nature of Mn 2+ compared with Mg 2+ and Ca 2+

25.  Ribozyme product extended by one nucleotide using α-32P-cordycepin (3- deoxyATP)  Digested with Ribonuclease T2 to reduce all end labeled material into nucleoside 3’ phosphates.  Carrier RNA also included generated using 4S UTP instead of UTP 2-Dimensional TLC system

26. Ribozymes: 4S U Ribonuclease T2 Carrier RNA: RNA C G A 4S U RNA

27. 2-Dimensional TLC system

28.  Ribozymes of RNA world need to promote reactions involving small organic molecules.  Uracil is significantly smaller than the smallest known ribozyme substrate  Found catalytic RNA can specifically recognize and utilize 4S Ura and can promote glycosidic bond formation  Support ribozyme-based metabolic pathways in RNA world

29. Further work  This ribozyme only capable of using one substrate  Could attempt to generate catalytic sequence capable of using two small- molecule substrates