Transcript RNA-catalysed nucleotide synthesis

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