Lecture 11 & 12: RNA world

Question 1

What evidence is there in the ribosome to suggest RNA world?

Answer 1

At the peptidyl transferase region (the core), there is a high density of Mg2+ ions, and as you move outward, the concentration of protein seen increases.

Provides a snapshot of evolution from RNA to protein.

Question 2

What phylogenetic evidence is available for the RNA world hypothesis?

Answer 2

Isoleucyl tRNA synthetase, and Valyl tRNA synthetase are paralogues, meaning they existed before LUCA.

There must have been some mechanism that involved Isoleucine and Valine before LUCA

Question 3

What evidence is there within co-factors to suggest RNA world?

Answer 3

There are RNA components in many cofactors for no functional reason.

This suggests an RNA molecule was gradually transitioned into becoming protein, however still remained as a small irreplaceable motif.

Question 4

What molecule was central to pre-biotic chemistry?

Answer 4

Cyanide - through reduction and contact with UV light, it is able to from amines, glycine, and subsequently amino acid pre-cursors.

Question 5

What is the idea of an RNA replicase?

Answer 5

A catalytic RNA molecule that is able to use its 3 dimensional shape allow it to catalyse its own synthesis from biological building blocks that are available.

Question 6

What are the requirements for RNA replicases?

Answer 6

-Initiation with a complimentary RNA primer

-Ribozyme docking

-Substrate binding

-Substrate discrimination: bonding the right sequence

-Regiospecific bond formation

-Strand Separation

Question 7

What needs to be prevented against for a RNA replicase to be successful?

Answer 7

-Template folding

-Likely that catalyst is complimentary to template, so self-pairing and formation of duplex needs to be prevented

Question 8

What is the catalytic potential of RNA?

Answer 8

General Acid-Base catalysis - however, does not have neutral pKa functional groups

pi-pi stacking with nucleobases

Mg2+-phosphate backbone coordination

RNA-RNA positioning

Question 9

What is the one constant for ribozymes?

Answer 9

They only catalyse phophodiesters.

Question 10

What is the basic principle of directed evolution?

Answer 10

Library of random-sequence RNA (multiple sizes + shapes)

Selection for desired property

Recovery and amplification of active variants

Question 11

What are some methods of selection?

Answer 11

Selection for ligation and hydrolysis: Gel purify for molecules that longer/shorter

Selection for self-modification: Ask for catalysis of non-phosphodiester molecules

Question 12

What was the initial experiment to test for ligative ribozymes?

Answer 12

RNA sequence was attached to an RNA hairpin that positioned a triphosphate next to a hydroxyl.

Using a 5 prime tag for detection, a catalytic domain within the sequence was asked to ligate the two together.

Question 13

Explain the formation of a processivity domain.

Answer 13

By round 18 of ribozyme selection, selection for the catalysis of one specific nucleotide was achieved, resulting in the formation of a processivity domain.

However, this required a lot of Mg2+, which led to degradation of the RNA.

Question 14

Explain the catalytic mechanism of the Round 18 RNA polymerase ribozyme.

Answer 14

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

Question 15

Explain the minor groove interactions.

Answer 15

Ribozyme makes minor groove interactions using a 3 adenine stretch.

RNA minor groove has a series of universal hydrogen bond acceptors.
(2’ hydroxyls, 2 keto-groups of C+U, N-3s of A+G)

RNA complex can evolve to contact this series of hydrogen bond acceptors

Question 16

What is the selection step from the R18 ribozyme to tC19Z?

Answer 16

Selection for in-trans activity, not self modification.

Library of single RNA molecules on beads, asking ribozymes to extend primers on the beads.

Amplification, and selection via fluorescence (check to see which beads are bright).

Question 17

What is the solution that tC19Z employs to extend primers?

Answer 17

Develops a 5’ tag that attaches directly to the end of the template.

Although the yield is not great, this allows for a high amount of primer elongation.

Question 18

What was the method for selection of functional RNA?

Answer 18

Link ribozyme to a primer, and ask it to synthesize a functional RNA molecule.

Selection via function to bind to a target, and fish out active ribozymes.
(Round 24 ribozyme)

Question 19

What is the effect of Round 52 ribozyme binding on catalysis?

Answer 19

Binding of 3’ OH at -3 primer position very important for catalytic activity.

Binding of -1 primer position important as a deoxy group at the end of the primer reduces the nucleophilicity of the 3’ OH.

Question 20

What are the two major issues with replicating ribozymes?

Answer 20

Fidelity. It has an error rate of 4.3%. As a result, the products made have less activity.

Still cannot completely self replicate.

Question 21

What ribozyme is able to replicate secondary strucutre?

Answer 21

Triplet polymerase ribozyme (TPR). It does this by attaching trinucleotides to the template to prevent refolding.

Trinucleotide substrates are able to also cooperatively bind to the RNA and unwind it.

Question 22

Explain the fidelity problem with triplet polymerase ribozymes.

Answer 22

Trinucleotide mispairing is much harder to characterize than mononucleotide base pairing.

The error rate increased as we moved from the 1st to the 3rd position on the trinucleotide.

Selection was done by adding a deoxy group to wobble trinucleotides, which would prevent elongation.

Question 23

Explain the structure of TPR.

Answer 23

Heterodimer made out of a 153n active domain, and 135n inactive domain.

Inactive domain stretches a single stranded sequence on the TPR that interacts with minor grooves on RNA.

Question 24

What environmental conditions do Ribozymes prefer?

Answer 24

The eutectic phase of water-ice.

The lower temperatures stabilize the RNA backbone

Question 25

How does strand separation and prevention of reannealing occur in TPR?

Answer 25

Acid used for strand separation. Protonation of A and C ruins base pairing.

Trinucleotides then coat the separate strands to prevent reannealing.

Question 26

Smallest ribozyme discovered that can provide the same activity?

Answer 26

QT45 (45n), not as fast.

Question 27

What are the 4 things required for protein coding mechanisms?

Answer 27

1. Message
2. Code
3. Peptidyl Transferase activity
4. Decoding function

Question 28

What are expansion segments?

Answer 28

New sequence emerging within a pre-existing secondary structure, without changing the pre-existing structure.

These can be used to extrapolate backwards to understand the development of the ribosome.

Question 29

What are A-minor interactions?

Answer 29

They are a directional interaction that allow an element on the ribosome interact with the pre-existing duplex.

Directional interaction allow us to deduce which structures emerged first.

Question 30

What are the first elements to emerge within the Small Ribosomal Subunit (SRU)?

Answer 30

Within the SRU the first element to emerge was the decoding center.

After that some structural elements, and then regions of interaction with the Large Ribosomal subunit.

Indication the LRU and SRU developed independently before coming together.

Question 31

What are the first elements to emerge within the Large Ribosomal Subunit (LRU)?

Answer 31

Within the LRU the first element to emerge was the peptidyl transferase center.

After that the exit tunnel, and then regions of interaction with the Small Ribosomal subunit.

Indication the LRU and SRU developed independently before coming together.

Question 32

What is the theory for where the SRU emerged from?

Answer 32

Due to its similarity with the TPR, it likely emerged from some RNA replicase ribozyme.

Question 33

What evidence is there to suggest a simpler amino acid code?

Answer 33

-Some current amino acids have a very complex biosynthesis, could have developed after protein

-Quite a few amino acids can be classified by family boxes: Where the third base of the codon does not matter. (only the first 2)

-Some amino acids were chemically prebiotically unavailable

-Synthetase classes may have needed to adapt to family box amino acids.

Question 34

How would you invent functions in de novo proteins?

Answer 34

1.Transcription of DNA molecule

2. Modification with puromycin (translational inhibitor).

3.Translate the mRNA, however, due to the puromycin, the protein remains attached to the mRNA

4. Selection for function can then take place
5. cDNA formed by reverse transcription can then by amplified by PCR.

Question 35

How can peptides benefit ribozymes?

Answer 35

A polylysine molecule enhances the activity of RNA polymerase ribozymes. (The interaction of the positive side chain with RNA stabilizes the complex).

This is particularly low at low Mg2+ conditions, so higher activity is possible, without degradation.