bioch430 LEC 2 NOTESv2

The RNA World Hypothesis proposes that early life used RNA as both the genetic material and as catalysts (ribozymes), so RNA carried information and performed biochemical reactions.

Tom Cech discovered self‑splicing Group I introns in Tetrahymena and Sidney Altman discovered RNase P activity in E. coli — key demonstrations that RNA can be catalytic.

Catalytic RNA enhances reaction rates and shows turnover (acts on many substrates), while autocatalytic RNA acts on itself to promote a reaction on the same molecule.

Small: hammerhead, hepatitis delta, hairpin. Medium: Group I, Group II, RNase P. Large: ribosome, spliceosome.

A catalyst lowers the activation energy by stabilizing the transition state, providing an alternative lower‑energy reaction pathway and thereby greatly accelerating reaction rates.

Turnover is how many catalytic cycles the catalyst performs per unit time (e.g., per minute); catalysts are not consumed by the reaction.

By binding and stabilizing different intermediates or transition states, catalysts may enable an alternative pathway with a lower energy barrier than the uncatalyzed route.

Pauling proposed that enzymes bind substrates in a conformation resembling the transition state, stabilizing it and lowering activation energy — a general principle of catalysis.

Two sequential transesterifications: (1) an external guanosine nucleophile attacks the 5' splice site; (2) the newly freed 5' exon attacks the 3' splice site to ligate exons and excise the intron.

He cloned the corresponding DNA into E. coli and showed intron excision and ligation depended only on the RNA sequence/folding, not on host proteins, demonstrating RNA‑intrinsic activity.

Because the precursor RNA autocatalyzes its own splicing during purification; the molecule itself is active and converts to product, making isolation of the unreacted precursor difficult.

The intron folds into a specific tertiary structure that positions reactants precisely, ensuring accurate transesterifications that yield the functional RNA product.

Phosphorothioate substitution makes the phosphate center chiral (Rp/Sp). Experiments showed Rp at the 5' splice site gives Sp in product (inversion), consistent with an SN2‑like displacement mechanism.

Inversion is consistent with a single‑step nucleophilic displacement (in‑line attack/SN2‑type) and supports the idea of a specific active site that enforces geometry.

Substituting sulfur creates a chiral phosphorothioate center (Rp/Sp) because sulfur differs from oxygen; this allows stereochemical probing of reaction mechanisms.

It indicates the active site is stereospecific—only the correct stereochemical configuration fits and can be processed, showing selectivity like protein enzymes.

The first step of splicing fails, but the reaction can be rescued by adding the nucleoside 2‑aminopurine (2AP), indicating specific interactions at that site.

2AP can form compatible interactions across the mutated base pair that mimic guanosine binding, enabling the first cleavage step in the mutant intron.

Arginine can competitively inhibit the wild‑type reaction by mimicking guanosine interactions; citrulline inhibits the mutant +2AP system—both act as competitive inhibitors showing specificity at the binding pocket.

They demonstrate the intron contains a specific G‑binding pocket that recognizes the guanosine nucleophile, behaving similarly to a protein enzyme’s substrate‑binding site.

By designing the intron so the 5' exon (substrate) is a separate oligonucleotide; the intron then folds to cleave that substrate repeatedly — converting an autocatalyst (cis) into a catalyst (trans).

To study catalytic properties like protein enzymes, to create site‑specific RNA cleavage tools, and to explore therapeutic targeting of RNAs.

Product inhibition and strong substrate/product binding (they look similar) reduce turnover; stability and specificity in cellular contexts are additional challenges.

In vitro site‑specific RNA cleavage and therapeutic targeting of viral RNAs; promising in concept but challenging in vivo.

Compensatory mutations (covariation) where one side of a base pair changes and the complementary side changes accordingly indicate conserved base pairing and help infer secondary structure.

Conserved regions are likely functionally or structurally important; variable regions are less constrained and may be peripheral or permissive to mutation.

P4–P6 is a folded domain whose X‑ray structure confirmed phylogenetic predictions; it shows how distal helices pack and that Mg2+ ions help stabilize the tertiary fold.

By biochemical probing (e.g., footprinting, mutagenesis) and structural determination (X‑ray crystallography) which confirm predicted pairings and tertiary contacts.

Jennifer Doudna solved the P4–P6 X‑ray structure, showing duplex regions fold back on themselves and revealing ordered Mg2+ ions at the interface that stabilize the fold.

Mg2+ neutralizes negative phosphate backbone charges and coordinates to specific sites, enabling compact tertiary folding and stabilizing active conformations.

A small, self‑cleaving RNA motif (named for its T‑shaped fold) that catalyzes site‑specific backbone cleavage when its strands assemble into the active architecture.

An in‑line attack: the 2'‑OH nucleophile must be collinear with the scissile phosphate and the leaving group — proper backbone/phosphate geometry is required.

The initial structure included a deoxy substitution at the cleavage site (removing the 2'‑OH nucleophile) and captured an inactive backbone conformation; larger constructs later revealed the active arrangement. p.104-107

Tertiary interactions toggle the ribozyme into an active conformation that aligns reactive groups; high salt and counterions can stabilize the fold, and metals may assist structurally though not always catalytically essential.

A highly conserved cytosine residue proximal to the cleavage site is essential, implicating a role for base‑mediated acid/base chemistry.

RNase A uses two conserved histidines; one acts as a general base to deprotonate the 2'‑OH nucleophile, the other as a general acid to protonate the leaving group — histidine pKa (~6.5) suits acid/base chemistry near neutral pH.

Yes — certain nucleobases (e.g., cytosine, adenosine) can be protonated/deprotonated in active sites and participate in acid/base catalysis similar to amino acid side chains.

X‑ray structures showed a fold distinct from other ribozymes but performing the same chemistry, illustrating the diversity of RNA architectures that can achieve similar catalytic outcomes.