L11 & 12 : RNA - First Darwinian molecule Flashcards
Ribosomal evidence for RNA world?
Peptidyl transferase centre (core of ribosome) is considered ribozyme
No protein residues within 20 A
Very high density of Mg2+ ions
Phylogenetic evidence for RNA world?
Isoleucyl and valyl tRNA synthetases are paralogues so can infer they diverged before LUCA
Cofactor evidence for RNA world?
Abundance of cofactors with RNA components for no functional reason.
RNA component gradually replaced with protein, though remained as small irreplaceable motif
What molecule was central to prebiotic chemistry?
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
What is a RNA replicase ribozyme?
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
What are the requirements for RNA self-replication?
- Initiation of synthesis
- General ribozyme docking to template
- Substrate binding and discrimination
- Regiospecific bond formation (3’-5’ phosphodiester)
- Strand separation after replication
What challenges are there for ribozymes?
Template folding - must prevent premature folding of ss sequence which may block progress
Self-pairing - Catalyst is complementary to template
What is the catalytic potential of RNA?
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)
What are classic ribozyme examples?
Nucleolytic ribozymes
Group I intron
Group II intron
RNaseP
What is the basic principle for directed evolution?
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)
What are some methods of selection?
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)
Evolution of RNA ligase activity?
Polymerising mononucleotide triphosphates
Elongate RNA molecule iteratively over and over
Analogous to ligation reaction
What was the first selection for ribozyme?
Random sequence region attached to RNA hairpin, positioning RNA phosphate next to hydroxyl
Using 5’ tag to ligate domains, giving range of catalytic domains
Explain the formation of processivity domain?
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
What is Round 18 Pol Ribozyme catalytic mechanism?
Template orientation
Triphosphate on incoming nucleotide interacts with Mg2+ to position pyrophosphate leaving group in line with attacking hydroxyl
What are the minor groove interactions?
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
Formation of processive polymerase ribozyme?
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
Formation of functional Round 24 ribozyme? (aptamer synthesis - RiboPCR)
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
What is Round 52 Ribozyme catalytic mechanism?
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
What are the current problems with replicating ribozymes?
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)
How does Triplet Pol Ribozyme synthesise RNA?
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
Substrate discrimination problem and selection pressure against misincorporation?
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
Explain the structure of TPR from cryo-SEM model
Heterodimer of 153nt active and 135nt accessory (inactive) domain
Inactive forms interactions with minor grooves of RNA, facilitating primer template binding
What were reaction conditions for ribozymes?
Eutectic phase of water-ice at -7 degrees
Lower temperatures stabilise RNA backbone
Ice crystal growth concentrates components
How were strands separated and reannealing prevented in TPR?
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
Smallest ribozyme with ability to self-synthesise?
QT45 with wobbly hairpin motif
Can a replicase survive?
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
Do RNA replicases require compartmentalisation?
Yes as may copy unrelated molecules, diluting function
Examples: model protocells, porous rock,
Where did RNA polymers come from?
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
How can RNA replicate RNA?
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)
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What is needed for coded protein synthesis emergence?
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
What is the molecular palaeontology of the ribosome and its development after LUCA?
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
What was the order of development in a ribosome?
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
How can development of ribosome structure be studied?
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
What conclusions can be made about early history of ribosome?
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
What features of the genetic code suggest a simpler form?
(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
What conclusions can be made about genetic code in prebiotic chem?
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)
What is the molecular palaeontology of peptides?
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
How can function be invented in de novo proteins?
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
How was function determined in non-coded peptides?
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
How do non-coded peptides relate to evolution of ribosome?
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
What is the peptidyl transferase reaction?
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
What is the role on 2’-OH on A2451 of rRNA?
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
What are the origins of aminoacylation?
Activated amino acid
Ribozyme catalysis
Local transfer
Genetic code?
Where did genetic code come from?
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
