1. RNA World Weaker hypothesis: There was a stage of evolution at when RNA molecules performed both genetic and catalytic roles. DNA later took over the genetic role and proteins took over the catalytic role. Stronger hypothesis: The RNA world arose de novo in the form of self replicating ribozymes. Almost certainly true The jury is still out Lectures based on: Joyce (2004) The antiquity of RNA-based evolution Nature 418: 214-221 Orgel (2004) Prebiotic chemistry and the origin of the RNA world. Crit. Rev. Biochem. Mol. Biol. 33: 99-123 Jeffares, Poole & Penny (1998) Relics from the RNA world. J. Mol. Evol. 46: 18-36.

2. Self-splicing introns. First RNA catalysts to be discovered. Tom Cech (1982). ‘RNA World’ term coined by Walter Gilbert (1986). RNA world idea originated in 60’s as a theoretical solution to the chicken and egg problem of DNA and proteins.

3. Hammerhead ribozyme Cleaves RNA at a specific point. Rolling circle mechanism of replication of virus-like RNAs in plants. Chops long strand into pieces. Example of an RNA catalyst

4. Chemistry required for RNA formation Prebiotic synthesis of sugars - Formose reaction – Butlerow (1861) Formaldehyde  Sugars (in aqueous solution) 2 CH 2 O + CH 2 OHCHO  2 CH 2 OHCHO formaldehyde glycoaldehyde Glycoaldehyde catalyses its own formation – autocatalytic Glycoaldehyde reacts to form a mixture of tetrose, pentose and hexose sugars.

5. Oro (1950s) showed plausible prebiotic synthesis of purines from HCN Many steps involved

6. Other steps in prebiotic synthesis Pyrimidines also ribose + base  nucleoside (weakest link) phosphorylation of nucleosides  nucleotides Conclusion from Orgel (2004) “no convincing prebiotic total synthesis of nucleotides” “many individual steps have been demonstrated” “few reactions give high yields” “complex mixtures of products”

7. Clutter of RNA synthesis (Joyce) Why is this particular set of monomers used for nucleic acids? How is this set synthesized specifically? Where is the chemistry occurring? Earth, or space? Hydrothermal vents?

8. Polymerization of nucleotides. Must be ‘activated’ - polymerization is uphill thermodynamically. ++  In vivo synthesis of RNA works like this – from nucleoside triphosphates. This found to be too slow in lab. Other activating groups were used. Oligomers up to 16mers formed using Pb++ catalyst. Up to 40mers formed using Montmorillonite clay catalyst (Ferris) Don’t know how monomers were activated prebiotically. Template directed synthesis of complementary strand possible from many oligomers.

9. An RNA organism must have had a metabolism. Hypothetical pathway for RNA catalyzed RNA synthesis (Joyce) Synthesis of nucleosides Phosphorylation Generation of NTPs Creation of activated nucleotides Stepwise polymerization

10. Can we synthesize a replicase ribozyme? Johnston et al. (2001) RNA-dependent RNA polymerase. General – should work on any sequence In vitro selection from large pool of random sequences Black – ribozyme Red – template Orange – primer Blue – randomized part In A, template is paired to ribozyme In C, it is not. Blue domain recognizes non- specific features of the template/primer. Templates extended by up to 14 extra nucleotides. Limited by ribozyme stability in solution. Fidelity 96.7% (see error threshold theory)

11. RNA Are there alternatives to RNA? a – Threose Nucleic Acid – TNA c – Glycerol derived nucleic acid b – Peptide nucleic acid – PNA d – Pyranosyl RNA RNA hybridizes with other nucleic acids. Information is not lost. DNA-RNA hybrids  DNA takes over at end of RNA world. Maybe TNA or PNA preceded the RNA world. Information passed to RNA. Would need to show that the alternative was easier to synthesize than RNA.

12. Two scenarios from Segré & Lancet (2000) A – RNA first (strong RNA world hypothesis) B – Lipids first (lipid world hypothesis – compositional genomes – metabolism without genes)

13. When was the RNA world? What preceded RNA? Another polymer? Metabolism only? Dating of rocks and meteorites Last ocean- vaporizing impact. Lunar craters Isotopic evidence for life (?) Microfossil evidence (?) Stromatolites. Phylogenetic methods (divergence after LUCA)

14. Why do we believe there was an RNA world (weaker hypothesis) ? Translation depends on RNA: mRNA supplies the information for protein synthesis. Active ingredient of the ribosome is rRNA – 3d structures show site of peptidyl transferase reaction. Proteins probably added as a late addition to the ribosome. tRNAs also essential for translation. rRNA and tRNA must be relics of the RNA world. Which other RNAs might be relics? Jeffares (1998) catalytic RNA more likely to be ancient than information-carrying RNA (newly evolved metabolic functions would be proteins) ubiquitous (all domains of life – prior to LUCA) central to metabolism

15. Tendency: RNA  RNP (ribonulceoprotein)  protein In early RNP particles, proteins act as cofactors to improve RNA catalysts. Eventually proteins replace RNA in most functions. Need genetic code to specify synthesis of long proteins Probable relic RNAs: Ribonuclease P – RNaseP (is a RNP) – tRNA processing snoRNAs – small nucleolar RNAs – rRNA processing (methylation of bases in specific positions on rRNA) snRNAs – small nuclear RNAs – mRNA processing in eukaryotes - spliceosome – U1, U2, U5 etc. several other possible candidates. Estimated genome of the last riboorganism – at least 10-15 kbp probably double stranded RNA genome (i.e. DNA came last, after RNA and proteins). Possibly several copies each of small linear chromosomes.