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RNA-catalysed nucleotide synthesis Peter J. Unrau & David P. Bartel

RNA-catalysed nucleotide synthesis Peter J. Unrau & David P. Bartel. Pamela Lussier Biochemistry 4000/5000. ‘RNA World’ Hypothesis. Hypothetical stage in origin of life on Earth.

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RNA-catalysed nucleotide synthesis Peter J. Unrau & David P. Bartel

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  1. RNA-catalysed nucleotide synthesisPeter 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 Nucleotide Pyrimidine Base

  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 SN1 reaction mechanism • Uracil is significantly smaller than the smallest ribozyme substrate.

  8. Pre-Adenylylation bypasses the specificity for donor substrate of T4 RNA ligase Figure 2 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. Ribozyme activity Triangle = uncatalyzed reaction rate After 4 rounds = ribozyme activity readily detected Round 4-6 = error prone PCR amplification Round 7-10 = decreasing the 4SUra concentration and decreasing the incubation time

  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 4SUra 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 • KM = Michaelis constant. Equal to the [S] at which the reaction rate is ½(Vmax). • E + S ES P + E k1 k2 K-1

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

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

  19. High Specificity for 4SUra • 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 4SUra 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 SN2 character

  23. Cofactors? • All three ribozyme families required divalent cations for activity. • Each round Mg2+ , Mn2+ and Ca2+ provided. • Ca2+ dispensable for all families • All preferred Mg2+ over Mn2+

  24. Family A did not need Mn2+ (twofold decrease in activity in absence of) • Family B and C require Mn2+, with the presence of 25mM Mg2+ reaching a plateau at 1mM Mn2+ • Family B ribozyme did not require for stimulating pRpp hydrolysis – Mn2+ has a role in binding or proper orientation of the 4SUra consistent with the thiophilic nature of Mn2+ compared with Mg2+ and Ca2+

  25. 2-Dimensional TLC system • 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 4SUTP instead of UTP

  26. Ribozymes: Ribonuclease T2 RNA 4SU 3'-deoxyATP Carrier RNA: RNA 3'-deoxyATP C RNA G 3'-deoxyATP RNA A 3'-deoxyATP RNA 3'-deoxyATP 4SU

  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 4SUra 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

  30. Questions?

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