RNAcatalysed nucleotide synthesis

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RNA-catalysed nucleotide synthesisPeter J. Unrau
David P. Bartel
Pamela Lussier Biochemistry 4000/5000
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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

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RNA-based life synthesized RNA from precursors
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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.

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Modern Metabolism
Activated Ribose
Pyrimidine Nucleotide
Pyrimidine Base
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• 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.

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• Unique to known RNA-catalysis
• Occurs by SN1 reaction mechanism
• Uracil is significantly smaller than the smallest
  ribozyme substrate.

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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
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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

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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
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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
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• 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

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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
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• Enzymes Kinetic parameters provide a measure of
  its catalytic efficiency
• Kcat Vmax/ET
• Number of rxn processes each active site
  catalyzes per unit time
• When SltltKm, little ES is formed
• E ET so equation below can reduce to a
  second order rate equation
• Vo k2ES (k2ETS)/(KM S)
• Can become
• Vo (Kcat/Km)ES

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• 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

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Isolates from each family promoted nucleotide
formation up to 107 times greater than upper
bound on uncatalysed reaction rate.
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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
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• 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
  doesnt aggregate of affect metal-ion
  availability.

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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.

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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?

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• 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.

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• RNA could have new strategy to promote glycosidic
  bond formation by stabilizing TS with more SN2
  character

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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

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• 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

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2-Dimensional TLC system

• Ribozyme product extended by one nucleotide using
  a-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

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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
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2-Dimensional TLC system
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• 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

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Further work

• This ribozyme only capable of using one substrate
• Could attempt to generate catalytic sequence
  capable of using two small-molecule substrates

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Questions?