Evidence/Exploting the RNA World Flashcards
What is the tree of life?
Shows how the different kingdoms are related - where modern biochemistry came from
It was an unknown what happened at the start - an unknown common ancestor - luca
Central Dogma:
DNA makes RNA that makes proteins
What do eukaryotic genes do?
They break simple co-linearity
When people began to sequence genes we realised their were introns that are larger than most of the exons
A new concept; splicing of exons, removal of introns
What experiments were conducted to understand how splicing works?
Two macromolecular machines were analysed
Tom Cech – self-splicing introns
He worked on a gene from a Tetrahymena - he knew one of the genes was spliced
He blended the Tetrahymena, fractionated the extract to work out what was responsible for the splicing of the gene
He found the splicing reaction was dependent on two components:
A guanine nucleotide - responsible from nucleophilic attack on some phosphodiester backbones
Divalent metal ion (Mg2+) - sets up the conformation of the RNA
Sidney Altman - RNAseP (enzyme that matures tRNA)
RNAseP consists of two components a protein part and RNA part
The dogma at the time suggested the protein would be the enzyme
He purified it and fractionated away the protein/RNA before doing an assay
He found the RNA part was maturing the tRNA the protein was just stabilising the RNA
Therefore RNA is catalytically active
How does the group I intron ribozyme mechanism work?
Standard nucleophilic attack from the guanine nucleotide forms the spliced exons
There is also an internalised nucleophilic attack from the guanine in the intron to form a cyclised intron
The RNA shows the catalytic ability of an enzyme
What is the structure of the intron ribozyme?
We can see that looking at longer RNA molecules they start to look more like proteins
They have Watson-Crick base pairings, bends, folding, a protein architecture
What are the roles of divalent metal ions in these splicing mechanisms?
Acid-base catalysis
Two-metal ion catalysis
How was the self-replicating RNA ribozyme discovered?
The Miller-Urey Experiment
CH4, NH3, H2O, H2 - sparked - condensed - heated (in a cycle)
They mimicked the atmospheric dynamics at the time
They found only ribose sugars - not deoxyribose = RNA molecules
Therefore we suspect that a prebiotic soup on Earth could have contained the chemical precursors of biopolymers, amino acids and nucleotide bases
Describe some extreme environments?
Black smokers - on the ridges of the tectonic plates, 100 degrees smoke
Around these we find archaea, extremophiles that are living there - very hard to culture in the lab
Mono-lake - NaCl2, very salty
We find extremophiles that thrive in the high salt of the lake - one has a purple membrane (uses metabolism similar to a plant)
Hot spring geyser - liquid in the middle, elemental sulphur is deposited, beyond that are extremophiles living in the hot run off the geyser
We use Taq polymerase in PCR which was extracted from an extremophile
What is significant about modern enzyme cofactors?
They are ribonucleotide derivatives
Amino acids will naturally react but just very slowly (without a ribosome)
Therefore pre-biology is a lot slower without enzymes - there’s no hurry
Ancient RNA cofactors that have not been replaced by amino acids
e.g. Acetyl-CoA
Describe the probability that random polymers produce catalysts?
• Modern day Urey & Miller experiments result in short polymers of polypeptide and oligonucleotides
Cech’s self-splicing intron is 100s of nucleotides long that is very improbable
People started to search for short RNA catalysts
What are viroids?
Start with a viroid - a self replicating RNA molecule, a potent pathogen to plants
Viroids do not code for any gene products (proteins) but are short (<200 nts) and can be deadly, e.g. cadang, cadang
It essentially disrupts the normally workings of the plant
Has a host RNA polymerase - it is persuaded to roll around the RNA - producing a continuous product - forming multimers of the basic genome being produced
The multimeric product has to be cleaved at particular sites using RNA catalysis - this is present in the viroid
The self-cleavage reaction is completed by a hammerhead ribozyme - the scissile bond and the Mg2+ will produce the cleavage
Describe ribonucleotide reductase?
Needed to help make 2’deoxyribose
Ribonucleotides are made first and subsequently converted into 2’ deoxynucleotides in a highly conserved reaction
Therefore they were around before the 2’deoxyribonucleotide reductases
= maybe RNA did come first
Descibe the ribosome?
The ribosome is mostly RNA decorated by ribosomal proteins
The ribosomal proteins play little role - mainly structural
There are no proteins near the peptidyl transferase site so the ribosome is an RNA enzyme, a ribozyme
The existence of RNA enzymes, ribozymes, like the modern ribosome, suggests that life may have evolved via an RNA World, into an RNA-Protein World that later discovered DNA as a way of storing lots of genetic information stably
What is the RNA world hypothesis?
A new Central Dogma:
RNA - chemically unstable but capable of folding into complex 3D shapes and producing active sites including for self-replication in a prebiotic soup
Molecular natural selection then drives the production of more efficient catalysts
Eventually an association with basic amino acids and peptides could stabilise these early replicators creating an RNA-Protein World
This makes
Protein - fold into complex shapes and their more diverse chemical side chains allow a wider range of catalysis to occur
RNA-Protein makes
DNA - a dull molecule chemically but whose structure is better for storing large amounts of genetic information
This is difficult to prove
But if you could see a different tree of life away from our planet could prove this is true
What do the catalytic properties of RNAs depend on?
- The fact that they contain uridine not thymidine
- The fact that they contain 2´ribose
- The fact that they fold spontaneously into complex 3D structures
- Their base pairing
- The fact that they can bind metal ions
This shows how RNA can do this and not DNA - due to the different chemistry
