RNA-catalysed RNA-catalysed nucleotide synthesisnucleotide synthesis

Peter J. Unrau & David P. BartelPeter J. Unrau & David P. Bartel

Pamela Lussier

Biochemistry 4000/5000

‘‘RNA World’ HypothesisRNA World’ Hypothesis Hypothetical stage in origin of life on Hypothetical stage in origin of life on

Earth.Earth. Proposes that early life developed by Proposes that early life developed by

making use of RNA molecules to store making use of RNA molecules to store information (DNA) and catalyze reactions information (DNA) and catalyze reactions (proteins)(proteins)

Thought that nucleotides constituting Thought that nucleotides constituting RNA were scarce on early EarthRNA were scarce on early Earth

RNA-based life synthesized RNA-based life synthesized RNA from precursorsRNA from precursors

RNA nucleotide synthesisRNA nucleotide synthesis Prebiotic synthesis Prebiotic synthesis

routes previously routes previously proposed for sugars, proposed for sugars, sugar phosphates, and sugar phosphates, and the four RNA bases.the four RNA bases.

Still a Challenge – Still a Challenge – coupling the molecules coupling the molecules into nucleotides.into nucleotides.

Modern MetabolismModern Metabolism

Activated Ribose

Pyrimidine Base

Pyrimidine Nucleotide

Release of pyrophosphate from activated Release of pyrophosphate from activated ribose causes nucleophilic attack on ribose causes nucleophilic attack on carboncarbon

Metabolic pathway forms both Metabolic pathway forms both nucleotides and amino acids tryptophan nucleotides and amino acids tryptophan and histidine in modern metabolismand histidine in modern metabolism

This mechanism is absent from known This mechanism is absent from known ribozyme reactions.ribozyme reactions.

Unique to known RNA-catalysis:Unique to known RNA-catalysis: Occurs by SOccurs by SN1N1 reaction mechanism reaction mechanism

Uracil is significantly smaller than the Uracil is significantly smaller than the smallest ribozyme substrate.smallest ribozyme substrate.

Figure 2 Pre-Adenylylation bypasses the specificity for donor substrate of T4 RNA ligaseThione 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 Steps for in vitroin vitro selectionselection pRpp attatched to 3’ end of pool RNApRpp attatched to 3’ end of pool RNA RNA-pRpp incubated with a 4-thiouracil RNA-pRpp incubated with a 4-thiouracil

(uracil analogue)(uracil analogue) RNA attached to newly synthesized RNA attached to newly synthesized

nucleotide 4-thiouridine were enriched, nucleotide 4-thiouridine were enriched, amplifiedamplified

Process of selection-amplification againProcess 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 4SUra concentration and decreasing the incubation time

Ribozyme activityRibozyme activity

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 – radio-To detect uncatalyzed reaction – radio-labelled pRpp-derivatized oligonucleotide labelled pRpp-derivatized oligonucleotide was incubated with was incubated with 4S4SUra and reaction Ura and reaction mixture was resolved on AMP gelmixture was resolved on AMP gel

Result = nothing detectedResult = nothing detected Gel could detect rates as slow as 6 x 10Gel could detect rates as slow as 6 x 10^-^-

77 M M-1-1 min min-1-1

Michaelis-Menten KineticsMichaelis-Menten Kinetics KKMM = Michaelis constant. Equal to the [S] at = Michaelis constant. Equal to the [S] at

which the reaction rate is ½(Vwhich the reaction rate is ½(Vmaxmax).).

E + S E + S ES P + EES P + Ek1

K-1

k2

Enzyme’s Kinetic parameters provide a Enzyme’s Kinetic parameters provide a measure of its catalytic efficiencymeasure of its catalytic efficiency

Kcat = Vmax/[E]Kcat = Vmax/[E]TT

Number of rxn processes each active site Number of rxn processes each active site catalyzes per unit timecatalyzes per unit time

When [S]<<Km, little ES is formed When [S]<<Km, little ES is formed [E] ~[E][E] ~[E]TT so equation below can reduce to a second so equation below can reduce to a second

order rate equation:order rate equation:

Vo = k2[ES] = (k2[ET][S])/(KM + [S])Vo = k2[ES] = (k2[ET][S])/(KM + [S])Can become:Can become:Vo = (Kcat/Km)[E][S]Vo = (Kcat/Km)[E][S]

Kcat/Km is the second-order rate Kcat/Km is the second-order rate constant of enzymatic reactionconstant of enzymatic reaction

Varies with how often enzyme and Varies with how often enzyme and substrate encounter each othersubstrate encounter each other

So kcat/Km is measure of enzymes So kcat/Km is measure of enzymes catalytic efficiencycatalytic 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 4SUra

Above14 mM – cannot measure due to Above14 mM – cannot measure due to solubility constraints.solubility constraints.

Cannot discount possibility that Cannot discount possibility that 4S4SUra Ura was starting to occupy inhibitory site, was starting to occupy inhibitory site, rather than catalytic site.rather than catalytic site.

Linear behavior of family b and c suggest Linear behavior of family b and c suggest 4S4SUra doesn’t aggregate of affect metal-Ura doesn’t aggregate of affect metal-ion availability.ion availability.

High Specificity for High Specificity for 4S4SUraUra Incubated all three ribozymes with thio-Incubated all three ribozymes with thio-

substituted bases (2-thiouracil, 2,4-substituted bases (2-thiouracil, 2,4-thiouracil, 2-thiocytosine, 2-thiopyrimidine, thiouracil, 2-thiocytosine, 2-thiopyrimidine, 2-thiopyridine, and 5-carboxy-2-thiouracil)2-thiopyridine, and 5-carboxy-2-thiouracil)

No thio-containing product detected on No thio-containing product detected on AMP gel.AMP gel.

Jump back to ProteinsJump back to Proteins

Thought to catalyze rxn by stabilizing Thought to catalyze rxn by stabilizing oxocarbocation at the C1- carbon of reaction oxocarbocation at the C1- carbon of reaction centercenter

Challenge: avoiding hydrolysisChallenge: avoiding hydrolysis Can avoid by excluding water from active site, Can avoid by excluding water from active site,

and promoting carbocation formation only after and promoting carbocation formation only after conformational change conformational change

What about Ribozymes?What about Ribozymes?

Examine degree of Examine degree of hydrolysis of tethered hydrolysis of tethered pRpppRpp

Promoted hydrolysis Promoted hydrolysis 12-23 x faster than 12-23 x faster than uncatalysed uncatalysed hydrolysishydrolysis

Rates for Rates for 4S4SUra Ura formation were formation were ≥60 ≥60 times faster than times faster than rates of catalysed rates of catalysed hydrolysis.hydrolysis.

RNA could have new strategy to promote RNA could have new strategy to promote glycosidic bond formation by stabilizing TS glycosidic bond formation by stabilizing TS with more Swith more SN2N2 character character

Cofactors?Cofactors? All three ribozyme families required All three ribozyme families required

divalent cations for activity.divalent cations for activity. Each round MgEach round Mg2+2+ , Mn , Mn2+2+ and Ca and Ca2+2+

provided.provided. CaCa2+ 2+ dispensable for all familiesdispensable for all families All preferred MgAll preferred Mg2+2+ over Mn over Mn2+2+

Family A did not need MnFamily A did not need Mn2+ 2+ (twofold (twofold decrease in activity in absence of) decrease in activity in absence of)

Family B and C require MnFamily B and C require Mn2+2+, with the , with the presence of 25mM Mgpresence of 25mM Mg2+2+ reaching a reaching a plateau at 1mM Mnplateau at 1mM Mn2+2+

Family B ribozyme did not require for Family B ribozyme did not require for stimulating pRpp hydrolysis – Mnstimulating pRpp hydrolysis – Mn2+2+ has a has a role in binding or proper orientation of the role in binding or proper orientation of the 4S4SUra consistent with the thiophilic nature Ura consistent with the thiophilic nature of Mnof Mn2+2+ compared with Mg compared with Mg2+2+ and Ca and Ca2+2+

Ribozyme product extended by one Ribozyme product extended by one nucleotide using nucleotide using αα-32P-cordycepin (3--32P-cordycepin (3-deoxyATP)deoxyATP)

Digested with Ribonuclease T2 to reduce Digested with Ribonuclease T2 to reduce all end labeled material into nucleoside 3’ all end labeled material into nucleoside 3’ phosphates.phosphates.

Carrier RNA also included generated Carrier RNA also included generated using using 4S4SUTP instead of UTPUTP instead of UTP

2-Dimensional TLC 2-Dimensional TLC systemsystem

Ribozymes:

4SU

Ribonuclease T2

Carrier RNA:

RNA

C

G

A

4SU

RNA

RNA

RNA

RNA

2-Dimensional TLC 2-Dimensional TLC systemsystem

Ribozymes of RNA world need to Ribozymes of RNA world need to promote reactions involving small organic promote reactions involving small organic molecules.molecules.

Uracil is significantly smaller than the Uracil is significantly smaller than the smallest known ribozyme substrate smallest known ribozyme substrate

Found catalytic RNA can specifically Found catalytic RNA can specifically recognize and utilize recognize and utilize 4S4SUra and can Ura and can promote glycosidic bond formationpromote glycosidic bond formation

Support ribozyme-based metabolic Support ribozyme-based metabolic pathways in RNA world pathways in RNA world

Further workFurther work This ribozyme only capable of using one This ribozyme only capable of using one

substratesubstrate Could attempt to generate catalytic Could attempt to generate catalytic

sequence capable of using two small-sequence capable of using two small-molecule substratesmolecule substrates