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RNA world hypothesis SELEX
RNA Structure Prediction and Comparison
Session 14
Exkurs: The RNA world
Jens Reeder
Faculty of TechnologyBielefeld University
Summer, 2008
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Outline
1 RNA world hypothesis
2 SELEX
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
The central dogma of molecular biology
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Difficulties in the evolution of replication
For ease of replication genetic information must be storedone-dimensional/sequential
Protein-to-protein replication not observed today (and mostlikely not anticipated at all)
Coded replication (as we see it today) requires lots ofmacromolecular components: genetic storage, mRNA, transferRNAs (tRNAs), aminoacyl-tRNA synthetases, ribosome
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
A chicken-egg problem
What evolved first: DNA or protein?
DNA needs proteins to replicate.Proteins encoded in DNA.
Solution: RNA world?
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
RNA world hypothesis
First hints in 1960’s.
C. Woese (1967): “The Genetic Code”
Crick (1968): RNA preceeded DNA and protein as source oflife.
W. Gilbert (1986): “The RNA world”, after discovery of firstcatalytic RNAs
RNA both as information storage and catalyst poweringrudimentary cells.
Later, DNA took over the role as genetic storage due to itschemical stability
Proteins (enzymes) replaced ribozymes - more variability andefficiency
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
RNA Milestones - Nobel prize 1989
Cleavage of precursor tRNA5’ends
Self-splicing group I introns fromTetrahymena thermophila
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
RNA polymerase
Current record: 20 ntelongation
RNA replicase only a matterof time
Example RNA polymerase:A: RNA ligase (up to 6 nuc.)B: initial SELEX poolC: 10 rounds of SELEXD: 18 rounds of SELEX
(16 nucs elongation)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
The equivalent of sex
Self-splicing group I introns from Tetrahymena thermophila
Implying reversibility, two separated introns can act as atransposon → recombination
Major boost for evolution.
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
RNA regulation
Even a simple metabolism needs regulation.Currently we know of:
thermoregulation
regulation by duplex formation
regulation induced by metabolite binding
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
A closer look
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Why do riboswitches support the RNA world hypothesis?
RNA riboswitches for universally required basic metabolites
RNA riboswitches more widespread in bacteria than anyprotein-based genetic factor that senses metabolites
Sensing domains carry most higly conserved elements(reasonable since the metabolites have not changed)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
The ribosome is a ribozyme
Ribosome composed of several proteins and RNAs
Catalytic core clearly defined by RNAs, proteins stabilize(2001)
Peptide bond formation catalyzed by adenin residue
Hypothesis: primitive ribosome made solely of RNAs
The smoking gun that convinced most biologist that therereally has been an RNA world
Lots of other catalytic activies reported (see second part)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
RNA world remnants and newcomers
RNAs provide the template for telomerase activity
RNAs part of the spliceosome
RNA editing (guide RNAs)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Intermediate conclusion
In a world of RNA there is:
replication
catalysis
mutation on short and large scale
regulation
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Consequences and benefits
Explanation for the early life on earth.
Simulation of evolution in the wet-lab
(Nearly) complete knowledge of the chemical repertoire ofRNA
Artificial ribozymes designed for specific task(pharmaceuticals)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
A simple ribo-organism
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Facts against the hypothesis
Chemical conditions on primitive earth might have led to rapidchemical degradation of RNA
Only a relative “short” timespan (0.5 billion years) untilevolution of first primitive life (3.5 bill. y.)
Did another replicating, evolving molecule preceeded RNA aswell?
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Outline
1 RNA world hypothesis
2 SELEX
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Evolution on the fast track
SELEX (Systematic Evolution of Ligands by ExponentialEnrichment) or in-vitro selection
Independently developed by three groups in 1990
Methodical components: synthesis of randomoligonucleotides, amplification via PCR, and transcription andreverse-transcription of RNA
Iterative process that amplifies DNA/RNA sequences withspecific properties.
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
SELEX - process
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Theoretical considerations
Library size: 1015 sequences
Complete coverage of sequence space for n = 25(theoretically)
Rapid decrease for longer sequences (n=100: 10−46)
Use only partially random sequences or bias the nucleotidecomposition
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Aptamers for target binding
simple ions
small molecules (amino acids, nucleotides, cofactors)
peptides and proteins
Theophyllin-dependent aptamer discriminates against caffeinemore than 10.000 fold (caffein and theophylin differ by onlyone methyl group)
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
A tryptophane transporter
RNA 9: membraneaffinity
RNA 727-44:tryptophane aptamer
RNA 10: linker
RNA 10Trp: 0.5 trp/secturnover rate,
Comparable to slowprotein transporters
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Catalysis
RNA cleavage, ligation and synthesis
Diels-Alder reaction
N-glycosidic and peptide bond formation
Alkylation acyl-transfer
...
Jens Reeder Bielefeld University
RNA Structure SS 08
RNA world hypothesis SELEX
Notes
Artificially selected Ribozymes and riboswitches are usuallysmaller than their natural occuring counterparts
However, they are also less efficient and specific
A current goal in SELEX experiments is to find “an exit fromthe RNA world”, e.g. to proove the existence of all ribozymesneccessary for protein synthesis.
Jens Reeder Bielefeld University
RNA Structure SS 08
