L11 & 12 : RNA - First Darwinian molecule

Question 1

Ribosomal evidence for RNA world?

Answer 1

Peptidyl transferase centre (core of ribosome) is considered ribozyme
No protein residues within 20 A
Very high density of Mg2+ ions

Question 2

Phylogenetic evidence for RNA world?

Answer 2

Isoleucyl and valyl tRNA synthetases are paralogues so can infer they diverged before LUCA

Question 3

Cofactor evidence for RNA world?

Answer 3

Abundance of cofactors with RNA components for no functional reason.
RNA component gradually replaced with protein, though remained as small irreplaceable motif

Question 4

What molecule was central to prebiotic chemistry?

Answer 4

Cyanide

Through reduction with UV. light, it is able to form a whole network of products including about8-12 of the natural precursors to AAs in biology today

Question 5

What is a RNA replicase ribozyme?

Answer 5

Catalytic RNA molecule whose 3D shape allows it to catalyse its own synthesis from biological building blocks available in the environment
Through cycles of reproduction and mutation

Question 6

What are the requirements for RNA self-replication?

Answer 6

• Initiation of synthesis
• General ribozyme docking to template
• Substrate binding and discrimination
• Regiospecific bond formation (3’-5’ phosphodiester)
• Strand separation after replication

Question 7

What challenges are there for ribozymes?

Answer 7

Template folding - must prevent premature folding of ss sequence which may block progress

Self-pairing - Catalyst is complementary to template

Question 8

What is the catalytic potential of RNA?

Answer 8

General acid base catalysis, though no neutral pKa functional groups
Pi-pi stacking interactions with nucleobase
Mg2+ phosphate backbone coordination
RNA-RNA positioning (eg. Watson-Crick pairing, Hoosteen interactions)

Question 9

What are classic ribozyme examples?

Answer 9

Nucleolytic ribozymes
Group I intron
Group II intron
RNaseP

Question 10

What is the basic principle for directed evolution?

Answer 10

Start with library of random sequence RNA molecules
Apply selection pressure for desired property/activity
Recovered and rtPCR amplification of active variants
Repeat (may utilise mutagenesis for diversity)

Question 11

What are some methods of selection?

Answer 11

For ligation/hydrolysis:
Selection for modules with larger/smaller size

For self-modification:
Chemically modify one terminus with reactive moiety
Look for catalysis of non-phosphodiester molecules (eg. peptide bond/Michael reaction)

Question 12

Evolution of RNA ligase activity?

Answer 12

Polymerising mononucleotide triphosphates
Elongate RNA molecule iteratively over and over
Analogous to ligation reaction

Question 13

What was the first selection for ribozyme?

Answer 13

Random sequence region attached to RNA hairpin, positioning RNA phosphate next to hydroxyl
Using 5’ tag to ligate domains, giving range of catalytic domains

Question 14

Explain the formation of processivity domain?

Answer 14

Round 18 RNA polymerase ribozyme
Selection for catalysis of direct incorporation of a specific mono ribonucleotides
Requires a lot of Mg2+, leading to ribozyme RNA degradation

Question 15

What is Round 18 Pol Ribozyme catalytic mechanism?

Answer 15

Template orientation
Triphosphate on incoming nucleotide interacts with Mg2+ to position pyrophosphate leaving group in line with attacking hydroxyl

Question 16

What are the minor groove interactions?

Answer 16

Interactions of processivity domain made using 3 adenine stretch of RNA minor groove
Series of universal conserved H bond acceptors in minor groove
Contact with backbone allows potential to copy any sequence

Question 17

Formation of processive polymerase ribozyme?

Answer 17

Selection for in-trans activity
Short 5’ tag hybridised directly to primer 3’ ends of library of single RNA molecules on beads (increases local concentration)
Amplification and selection via fluorescence

Question 18

Formation of functional Round 24 ribozyme? (aptamer synthesis - RiboPCR)

Answer 18

Link ribozyme to primer and ask for synthesis of complete functional RNA
Select for aptamer function to bind target on beads
Ribozyme can now catalyse reaction similar to PCR

Question 19

What is Round 52 Ribozyme catalytic mechanism?

Answer 19

Original structure of ribozyme changes and refolds so no longer needs to hybridise + measurably higher affinity for template

Binding of 3’OH at -3 primer position and binding at -1 primer position important for catalysis

Question 20

What are the current problems with replicating ribozymes?

Answer 20

Fidelity - error rate of 4.2% so products have lower activity

Still cannot completely self-replicate - template secondary structure (could add oligos to bind and disrupt/prevent formation)

Question 21

How does Triplet Pol Ribozyme synthesise RNA?

Answer 21

Triplet oligonucleotides bind cooperatively to the template to prevent refolding
Trinucleotides bind and can provide substrates ready for ligation also.

Divides catalytic core - synthesis of segments and ligation activity to join segments

Question 22

Substrate discrimination problem and selection pressure against misincorporation?

Answer 22

Harder to discriminate incorrect trinucleotides than mononucleotides causing increase in error rate for 3rd position

Selected against by adding 3’ deoxy (rather than hydroxyl) to wobble trinucleotides, preventing further elongation

Question 23

Explain the structure of TPR from cryo-SEM model

Answer 23

Heterodimer of 153nt active and 135nt accessory (inactive) domain
Inactive forms interactions with minor grooves of RNA, facilitating primer template binding

Question 24

What were reaction conditions for ribozymes?

Answer 24

Eutectic phase of water-ice at -7 degrees
Lower temperatures stabilise RNA backbone
Ice crystal growth concentrates components

Question 25

How were strands separated and reannealing prevented in TPR?

Answer 25

Difficult to separate RNA duplex and rapid reannealing (faster than replication occurs) Strand separation using acid to break Watson-Crick pairing Repetitive cycles of acidification and heating, neutralising, flash freezing

Question 26

Smallest ribozyme with ability to self-synthesise?

Answer 26

QT45 with wobbly hairpin motif

Question 27

Can a replicase survive?

Answer 27

Self replication rate a Hydrolysis rate d Incorrect copy formation rate of 1-q (q=correct rate) Survival threshold: aQ > s Error threshold: a/a(from mutated replicase) > 1/Q If this fails, mutated species will outcompete original Error threshold is whether evolution promotes retention of phenotype or mutation

Question 28

Do RNA replicases require compartmentalisation?

Answer 28

Yes as may copy unrelated molecules, diluting function Examples: model protocells, porous rock,

Question 29

Where did RNA polymers come from?

Answer 29

Condensation from RNA building blocks and gradual lengthening Doesn't need to be sustainable but need opportunities Can be done using phosphorimidazolide: imidazole leaving group more reactive than pyrophosphate Only tends to work for G nucleotides - creates bridged imidazolium dinucleotide If this is incubated with RNA duplex get greatly accelerated non-enzymatic RNA synthesis Rna template is the enzyme

Question 30

How can RNA replicate RNA?

Answer 30

Recruit building blocks using W-C pairing (colocalisation of nucleophile and electrophile via template binding) Ensure correct substarte selection to accurately copy information (minor groove, danger of wobble pairing) Catalyse phosphodiester bond formation (position 3' OH near 5' P and align leaving group) Turn primer extension into RNA replication (using physiochemical cycles, preventing reannealing) Turn RNA replication into RNA evolution (must act on itself via compartmentalisation)

Question 31

NEXT LECTURE

Answer 31

NEXT LECTURE

Question 32

What is needed for coded protein synthesis emergence?

Answer 32

RNA message - when translated, gives phenotypic advantage Code - relationship between RNA sequence and AAs to be incorporated Peptidyl transferase activity - join building blocks to produce protein Decoding function

Question 33

What is the molecular palaeontology of the ribosome and its development after LUCA?

Answer 33

Conserved structure in centre of ribosome Elaborations of structure from prokaryote to eukaryotes Addition of new RNA sequences by expansion segments, without changing secondary structure already present

Question 34

What was the order of development in a ribosome?

Answer 34

By studying ribosomal secondary structure (directionality of A minor interactions) together with order of emergence, can get sense of order of development Within small subunit, decoding centre first emerged, followed by structural regions and interfaces for large subunit Within large subunit, PTC first emerged, followed by exit tunnel development and interfaces for small subunit Subunits developed independently before associating

Question 35

How can development of ribosome structure be studied?

Answer 35

Can extrapolate backwards to approx delineate development, sense of order of emergence Can look at how different elements interact with each other - A minor interactions have directionality allowing inference of evolutionary history

Question 36

What conclusions can be made about early history of ribosome?

Answer 36

Conserved structure of ribosome reveals history of accretion - Replacement of Mg2+ with peptides further out from active site, charting evolutionary emergence of non-coded (rRNA) and coded (ribosomal proteins) components in protein synthesis - Subunit interface arose after emergence of independent subunit functions (PTC + triplet recognition) - Hierarchy of protein conformation integrated with ribosome structure (unstructured, local b hairpins, globular domains) - Protuberances as sites of interactions with elongation factors appear later

Question 37

What features of the genetic code suggest a simpler form?

Answer 37

(Met, Lys, Trp) have complex biosynthesis so difficult to justify in prebiotic chem - post-development of coded protein synthesis? (Val, Gly, Ala) have family boxes where third base not necessary (Tyr, His) side chains prebiotically unavailable Some not present when synthetases arose

Question 38

What conclusions can be made about genetic code in prebiotic chem?

Answer 38

Don't need all 20 AAs to invent coded protein synthesis, need only partial correlation justify coded mechanism Genetic code reflects prebotic chemistry and CN-/UV/SO3- based reactions 9 AAs : Gly, Ala, Val, Ile, Leu, Ser, Thr, Pro, Arg Need only ~9 tRNAs and synthetases (rather than ~40 and 20)

Question 39

What is the molecular palaeontology of peptides?

Answer 39

Successfully folded protein domains rare in random sequence New folds emerge by repetition and decoration Studying abundance of structures throughout modern proteome, possible to identify some ancient candidate peptide folds At least 40 ancestral peptides proposed

Question 40

How can function be invented in de novo proteins?

Answer 40

Library of DNA molecules that are transcribed Modify 3' end with puromycin (translational inhibitor) mRNA library translated and proteins synthesised, which attach to the end of the mRNA Can generate library of mRNA molecules with proteins they encode In vtiro selection and recovery based on function, reverse transcription and rtPCR

Question 41

How was function determined in non-coded peptides?

Answer 41

Small peptides can reduce dependence of RNA world on Mg2+ ions (reduced RNA degradation) Replacing Mg2+ for RNA pol ribozymes Pol-L-lysine is positively charged so stabilises RNA structure and drives coacervate formation to boost ligation, changing coacervate properties Cys-rich peptides form catalytic FeS clusters to carry out functions that couldn't otherwise be achieved

Question 42

How do non-coded peptides relate to evolution of ribosome?

Answer 42

Some function possible in non-coded peptides of biased composition Possible motive for PTC evolution before coding, compensating for RNA functional weaknesses through: - positive charge - hydrophobic - FeS cluster binding Nature of first coded peptides remains unclear and requires studying RNA-peptide coevolution

Question 43

What is the peptidyl transferase reaction?

Answer 43

Thermodynamically favourable reaction forming amide bonds during translation Amine group of incoming AA attacks ester bond of growing peptide chain Problem: Forms transition sate with protonated amine Hydroxyl group formed has higher pKa than amine Solution: Needs general base catalyst to remove proton from amine so more reactive

Question 44

What is the role on 2'-OH on A2451 of rRNA?

Answer 44

Thought to act as proton shuttle by acting as general acid catalyst (donates proton to hydroxyl leaving group) and general base catalyst (removes proton from amine) This catalytic role of ribosomal RNA assisting in stabilising transition state supports idea of RNA world

Question 45

What are the origins of aminoacylation?

Answer 45

Activated amino acid Ribozyme catalysis Local transfer Genetic code?

Question 46

Where did genetic code come from?

Answer 46

Biological origin: aminoacylation originally served another function (stabilisation or facilitating RNA folding) Chemical origin: predisposed chemical/molecular relationship between AAs and specific RNA triplets As RNA-peptide systems co-evolved, specific attachment of AAs to RNA (aminoacylation) and ribosomes ability to catalyse peptidyl transferase reactions to form peptide bonds may have contributed to emergence of genetic code