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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. 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 RNA-based life synthesized RNA from precursors 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. Modern Metabolism Activated Ribose Pyrimidine Base Pyrimidine Nucleotide 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. Unique to known RNA-catalysis: Occurs by SN1 reaction mechanism Uracil is significantly smaller than the smallest ribozyme substrate. 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 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 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 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 To detect uncatalyzed reaction – radiolabelled 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 Michaelis-Menten Kinetics KM = Michaelis constant. Equal to the [S] at which the reaction rate is ½(Vmax). E+S k1 ES K-1 k2 P+E 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] 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 Isolates from each family promoted nucleotide formation up to 10^7 times greater than upper bound on uncatalysed reaction rate. Fits to a MichaelisMenten curve Do not display saturable behavior Suggests poorer binding to 4SUra Circle = Family A – a15 Square = Family B – b01 Diamond = Family C – c05 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 metalion availability. High Specificity for 4SUra Incubated all three ribozymes with thiosubstituted bases (2-thiouracil, 2,4thiouracil, 2-thiocytosine, 2-thiopyrimidine, 2-thiopyridine, and 5-carboxy-2-thiouracil) No thio-containing product detected on AMP gel. 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? 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. RNA could have new strategy to promote glycosidic bond formation by stabilizing TS with more SN2 character 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+ 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+ 2-Dimensional TLC system Ribozyme product extended by one nucleotide using α-32P-cordycepin (3deoxyATP) Digested with Ribonuclease T2 to reduce all end labeled material into nucleoside 3’ phosphates. Carrier RNA also included generated using 4SUTP instead of UTP Ribozymes: Ribonuclease T2 RNA 4SU Carrier RNA: RNA C RNA G RNA A RNA 4SU 2-Dimensional TLC system 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 Further work This ribozyme only capable of using one substrate Could attempt to generate catalytic sequence capable of using two smallmolecule substrates