Small RNA inside and outside the cell Memories on early evolution or recent developments? (Title by courtesy of Eberhard Neumann) Peter Schuster Institut für Theoretische Chemie, Universität Wien, Austria and The Santa Fe Institute, Santa Fe, New Mexico, USA 42. Winterseminar Klosters, 14.– 27.01.2007
Recent review article: Peter Schuster, Prediction of RNA secondary structures: From theory to models and real molecules Rep. Prog. Phys . 69 :1419-1477, 2006. Web-Page for further information: http://www.tbi.univie.ac.at/~pks
1. The exciting RNA story 2. Why is gene regulation so complex? 3. What small RNAs can achieve 4. Structures of small RNAs 5. Riboswitches and kinetic folding
1. The exciting RNA story 2. Why is gene regulation so complex? 3. What small RNAs can achieve 4. Structures of small RNAs 5. Riboswitches and kinetic folding
RNA as scaffold for supramolecular complexes RNA as catalyst Ribozyme ribosome ? ? ? ? ? RNA RNA is modified by epigenetic control RNA editing RNA The world as a precursor of DNA protein the current + biology RNA Alternative splicing of messenger RNA as carrier of genetic information RNA viruses and retroviruses RNA evolution in vitro Functions of RNA molecules
Jack W. Szostak. RNA gets a grip on translation. Nature 419 :890-891 (2002) Wade Winkler, Ali Nahvi, Ronald R. Breaker. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419 :952-956 (2002)
Stéphan Thore, Marc Leibundgut, Nenad Ban. Structure of eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 312 :1208-1211 (2006)
Joanna Owens. Riboswitching off bacterial growth. Nature Reviews /Drug Discovery 6 :23 (2007) K.F. Blount et al. Antibacterial lysine analogs that target lysine riboswitches. Nature Chem. Biol. 3, December (2006) Alexey G. Vitreschak, Dimitry A. Rodinov, Andrey A. Mironov, Mikhail S. Gelfand. Riboswitches: The oldest mechanism for the regulation of gene expression? TRENDS in Genetics 20 :44-50 (2004)
Nobel prize for medicine 2006 Andrew Z. Fire Stanford University Craig C. Mello University of Massachusetts Worcester Gene silencing by small interfering RNAs
1. The exciting RNA story 2. Why is gene regulation so complex? 3. What small RNAs can achieve 4. Structures of small RNAs 5. Riboswitches and kinetic folding
L.J. Croft, M.J. Lercher, M.J. Gagen, J.S. Mattick. Is prokaryotic complexity limited by accelerated growth in regulatory overhead? Genome Biology 5 :P2 (2003)
A model for genome duplication in yeast � 1 � 10 8 years ago 2 new genes out of 16 genes, sequence of genes largely modified Manolis Kellis, Bruce W. Birren, and Eric S. Lander. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae . Nature 428 : 617-624, 2004
1. The exciting RNA story 2. Why is gene regulation so complex? 3. What small RNAs can achieve 4. Structures of small RNAs 5. Riboswitches and kinetic folding
Evolution of RNA molecules based on Q β phage D.R.Mills, R.L.Peterson, S.Spiegelman, An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule . Proc.Natl.Acad.Sci.USA 58 (1967), 217-224 S.Spiegelman, An approach to the experimental analysis of precellular evolution . Quart.Rev.Biophys. 4 (1971), 213-253 C.K.Biebricher, Darwinian selection of self-replicating RNA molecules . Evolutionary Biology 16 (1983), 1-52 G.Bauer, H.Otten, J.S.McCaskill, Travelling waves of in vitro evolving RNA. Proc.Natl.Acad.Sci.USA 86 (1989), 7937-7941 C.K.Biebricher, W.C.Gardiner, Molecular evolution of RNA in vitro . Biophysical Chemistry 66 (1997), 179-192 G.Strunk, T.Ederhof, Machines for automated evolution experiments in vitro based on the serial transfer concept . Biophysical Chemistry 66 (1997), 193-202 F.Öhlenschlager, M.Eigen, 30 years later – A new approach to Sol Spiegelman‘s and Leslie Orgel‘s in vitro evolutionary studies . Orig.Life Evol.Biosph. 27 (1997), 437-457
Chemical kinetics of molecular evolution M. Eigen, P. Schuster, `The Hypercycle´, Springer-Verlag, Berlin 1979
Complementary replication is the simplest copying mechanism of RNA. Complementarity is determined by Watson-Crick base pairs: G � C and A = U
Replication rate constant : f k = � / [ � + � d S (k) ] � d S (k) = d H (S k ,S � ) Selection constraint : Population size, N = # RNA molecules, is controlled by the flow ≈ ± ( ) N t N N Mutation rate : p = 0.001 / site � replication The flowreactor as a device for studies of evolution in vitro and in silico
Quasispecies Uniform distribution 0.00 0.05 0.10 Error rate p = 1-q Quasispecies as a function of the replication accuracy q
Quasispecies The error threshold in replication
Target structure Simulation of the approach to a target structure with a population size of N=3000 RNAs
Evolutionary design of RNA molecules D.B.Bartel, J.W.Szostak, In vitro selection of RNA molecules that bind specific ligands . Nature 346 (1990), 818-822 C.Tuerk, L.Gold, SELEX - Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase . Science 249 (1990), 505-510 D.P.Bartel, J.W.Szostak, Isolation of new ribozymes from a large pool of random sequences . Science 261 (1993), 1411-1418 R.D.Jenison, S.C.Gill, A.Pardi, B.Poliski, High-resolution molecular discrimination by RNA . Science 263 (1994), 1425-1429 Y. Wang, R.R.Rando, Specific binding of aminoglycoside antibiotics to RNA . Chemistry & Biology 2 (1995), 281-290 Jiang, A. K. Suri, R. Fiala, D. J. Patel, Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex . Chemistry & Biology 4 (1997), 35-50
An example of ‘artificial selection’ with RNA molecules or ‘breeding’ of biomolecules
The SELEX technique for the evolutionary preparation of aptamers
Aptamer binding to aminoglycosid antibiotics: Structure of ligands Y. Wang, R.R.Rando, Specific binding of aminoglycoside antibiotics to RNA . Chemistry & Biology 2 (1995), 281-290
tobramycin -3’ 5’- G C A C G A U U U A C U A C A C U C G U C G G G G G C U U 5’- G C A C G A G G G U A RNA aptamer 3’- G C C G U C C A G U C A U C Formation of secondary structure of the tobramycin binding RNA aptamer with K D = 9 nM L. Jiang, A. K. Suri, R. Fiala, D. J. Patel, Saccharide-RNA recognition in an aminoglycoside antibiotic- RNA aptamer complex. Chemistry & Biology 4 :35-50 (1997)
The three-dimensional structure of the tobramycin aptamer complex L. Jiang, A. K. Suri, R. Fiala, D. J. Patel, Chemistry & Biology 4 :35-50 (1997)
Hammerhead ribozyme – The smallest RNA based catalyst H.W.Pley, K.M.Flaherty, D.B.McKay, Three dimensional structure of a hammerhead ribozyme . Nature 372 (1994), 68-74 W.G.Scott, J.T.Finch, A.Klug, The crystal structures of an all-RNA hammerhead ribozyme: A proposed mechanism for RNA catalytic cleavage . Cell 81 (1995), 991-1002 J.E.Wedekind, D.B.McKay, Crystallographic structures of the hammerhead ribozyme: Relationship to ribozyme folding and catalysis . Annu.Rev.Biophys.Biomol.Struct. 27 (1998), 475-502 G.E.Soukup, R.R.Breaker, Design of allosteric hammerhead ribozymes activated by ligand- induced structure stabilization . Structure 7 (1999), 783-791
Allosteric effectors: FMN = flavine mononucleotide H10 – H12 theophylline H14 Self-splicing allosteric ribozyme H13 theophylline Hammerhead ribozymes with allosteric effectors
A ribozyme switch E.A.Schultes, D.B.Bartel, Science 289 (2000), 448-452
Two ribozymes of chain lengths n = 88 nucleotides: An artificial ligase ( A ) and a natural cleavage ribozyme of hepatitis- � -virus ( B )
The sequence at the intersection : An RNA molecules which is 88 nucleotides long and can form both structures
Two neutral walks through sequence space with conservation of structure and catalytic activity
1. The exciting RNA story 2. Why is gene regulation so complex? 3. What small RNAs can achieve 4. Structures of small RNAs 5. Riboswitches and kinetic folding
GGCUAUCGUACGUUUACCCAAAAGUCUACGUUGGACCCAGGCAUUGGACG One error neighborhood – Surrounding of an RNA molecule in sequence and shape space
GGCUAUCGUACGUUUACCCAAAAGUCUACGUUGGACCCAGGCAUUGGACG One error neighborhood – Surrounding of an RNA molecule in sequence and shape space
GGCUAUCGUACGUUUACCCAAAAGUCUACGUUGGACCCAGGCAUUGGACG G G A U C U G A C CC C A GG G G C U UGGA A U C UACG U G U C A G U AAG UC U A U C C C AA One error neighborhood – Surrounding of an RNA molecule in sequence and shape space
G GGCUAUCGUACGUUUACCC AAAGUCUACGUUGGACCCAGGCAUUGGACG GGCUAUCGUACGUUUACCCAAAAGUCUACGUUGGACCCAGGCAUUGGACG G G A U C U G A C CC C A GG G U G U G C A U A C G U A A A A G G C U A C U A C G U U C G U A C A G A C A G C G G C G U A G U G U A C G U C A A U C U A C G G C A C G U G G A C A G G C U G U U A G C U UGGA A U C UACG U G U C A G U AAG UC U A U C C C AA One error neighborhood – Surrounding of an RNA molecule in sequence and shape space
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