Peter J. Unrau & David P. Bartel
Pamela Lussier
Biochemistry 4000/5000

 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

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

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 S
N1 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

 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

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

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 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

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

 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.

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

 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 metalion availability.

4S
 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.

 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 4S Ura 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 S
N2 character

 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+

 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+

 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 4S UTP instead of UTP

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

 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

 This ribozyme only capable of using one substrate
 Could attempt to generate catalytic sequence capable of using two smallmolecule substrates