RNA-världen: forntid i nutid med framtid. Leif A. Kirsebom

Transkript

1 RNA-världen: forntid i nutid med framtid Leif A. Kirsebom

2 Livets ursprung Virtanen & Kirsebom, NFRs årsbok 1997

3 Livets ursprung history of life Vad kom först?

4 Livets ursprung förutsättningar?

5 Centrala dogmat - Hur ser det ut idag Ribonucleotide reductase! Virtanen & Kirsebom, NFRs årsbok 1997

6 Genetiska koden

7 RNA genotyp och fenotyp Vad kom först? RNA possess both genotype - represented by a sequence that can be copied by a polymerase - and phenotype - represented by some functional trait that varies according to sequence.

8 RNA-världen (definition) 1. RNA-världens genetiska material var RNA 2. Basparning enl. Watson-Crick-modell 3. Genetiskt nedärvda proteiner deltog inte som katalysatorer Evidence?

9 "Molecular fossils" Molekylära fossiler: En idag förekommande struktur eller funktion som har ett gammalt ursprung och som ger oss en ledtråd om de tidigaste formerna av liv Struktur: basen adenin Funktion: reaktionsväg ribos (RNA) blir deoxyribos (DNA) ribosomen (RNA gör protein)

10 "Molecular fossils" Coenzyme e.g. NAD/ NADH; FAD Redox reactions/ energy Metabolites e.g. thiamin pyrophosphate, tetrahydrofolate, pyridoxal phosphate - Riboswitches Histidine only amino acid where the synthesis start with a nucleotide Nature 441, (2006) Adapted from Yarus M Cold Spring Harb Perspect Biol 2011;3:a003590

11 Små RNA molekyler:! hammarskallestrukturen är katalytisk! RNA genetiskt material RNA (viroids, virosoides, virus) RNA katalytiskt! Virtanen & Kirsebom, NFRs årsbok 1997

12 RNA katalytiskt Större RNase P och andra Virtanen & Kirsebom, NFRs årsbok 1997 RNase P Domains and Activity Splicing Telomerase Riboswitches Ribosomen etc Reiter et al. Nature 2010 Metal(II) joner!

13 RNase P - Domains and Activity

14 Virtanen & Kirsebom, NFRs årsbok 1997 RNA och proteinsyntes

15 trna, the Genetic code, the Adaptor Hypothesis (1954/55) and the RNA tie club Phe phenylalanine Phe-tRNA 5' 3' Phe D-loop T-loop Anticodon loop Pre-tRNA RNase P RNA (katalytisk) Protein synthesis Genome replication e.g. plant virus, RNA primer (bakterie replikation), retrovirus (HIV), telomers etc Synthesis of chlorophyll a (from eight δ-aminolevulinate)

16 "Molecular Mimicry" EF-Tu-tRNA EF-G Shape Electrostatics EMBO J 13, (1994) Czworkowski et al. EMBO J 19, (2000) Nissen, Kjeldgaard, Nyborg J Mol Biol 303, (2000) Laurberg et al.

17 RNA-världen: forntid i nutid med framtid Virtanen & Kirsebom, NFRs årsbok 1997

18 "Primordiala soppan" Darwin letter to his friend and supporter Joseph Hooker..that life may have taken place in a warm little pond, with all sorts of ammonia and phosphoric salts, lights, heat, electricity, etc. present, so that a proteine compound was chemically formed ready to undergo still more complex changes. Citation taken from RNA: Life s indispensable molecule J. Darnell 1. Små organiska föreningar ex.vis. formaldehyd, aminosyror, adenin, socker 2. Oorganiska föreningar, atomer, molekyler t ex metalljoner, mineraler, lera miller-urey-experiment blogs.nature.com Oparin (1928)

19 Evidence för en RNA-värld Byggstenar RNA ett måste "Pyrimidine ribonucleotides can be formed in a short sequence that bypasses free ribose and the nucleobases, and instead proceeds through arabinose amino-oxazoline and anhydronucleoside intermediates." Matthew W. Powner, Béatrice Gerland and John D. Sutherland Nature 459, (2009) nntps npp i R + P RP closed RP open RNA

20 Evidence för en RNA-värld Evolution in the test tube - SELEX Joyce. (2002), Nature 418, 214 Virtanen & Kirsebom, NFRs årsbok 1997

21 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens PCR Aptamer Klon ds DNA pool PCR RNA Transkription RT RNA Binding Selektion Anrikning RNA

22 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 PCR Aptamer Klon ds DNA pool PCR RNA Transkription RT RNA Binding Selektion Anrikning RNA

23 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 3 PCR Aptamer Klon ds DNA pool PCR RNA Transkription RT RNA Binding Selektion Anrikning RNA

24 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 3 PCR Aptamer Klon ds DNA pool PCR RNA Transkription RT 4 RNA Binding Selektion Anrikning RNA

25 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 3 PCR Aptamer Klon ds DNA pool PCR RNA 5 Transkription RT 4 RNA Binding Selektion Anrikning RNA

26 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 3 PCR 6 Aptamer Klon ds DNA pool PCR RNA 5 Transkription RT 4 RNA Binding Selektion Anrikning RNA

27 Evolution in the test tube - SELEX 1 Syntetisk DNA pool T7 promoter 5' 3' Konstant sekvens Slumpad sekvens Konstant sekvens 2 3 PCR 6 Aptamer Klon ds DNA pool PCR RNA 5 7 Transkription RT 4 RNA Binding Selektion Anrikning RNA

28 Evolution in the test tube - SELEX Searching for sequencing space for new functional RNA sequences. Altering the ratio active/inactive sequences. Complete coverage N = number of nucleotides randomized 25! Compared to for example N = 220, which equals variants!!!!! Impossible therefore...when longer randomized sequences are used it is a question of probability to score!!

29 Evolution in the test tube - SELEX RNA replicase/ transcriptase Binding of various small ligands e.g. ATP RNA catalyzed synthesis of nucleotide RNA catalyzed synthesis of nucleotide RNA cleavage RNA Joyce. (2002), Nature 418, 214

30 Copy RNA - RNA polymerases RNA based polymerase 40 kda (120 nts) Protein based polymerase 400 kda Active site/ Catalytic centre nntps npp i R + P RP closed RP open RNA Shechner and Bartel (2011) Nat Struc Mol Biol 18, 1036 Vassylyev et al. (2002) Nature 417, 712

31 Virtanen & Kirsebom, NFRs årsbok 1997 RNA och proteinsyntes

32 Aminoacylering av RNA med RNA Yarus et al., Proc Natl Acad Sci USA (2010) Substrate 3' 5' G C C U + Produkt 3' C G G U G RNA katalysator 5' fold down

33 RNA mediated cleavage of RNA Identification of a complex consisting of seven nucleotides that results in RNA mediated cleavage in the presence of Mn 2+ Substrate 3' 5' G A A A + Mn2+ Produkt 3' U U U RNA katalysator 5' Kazakov and Altman Proc Natl Acad Sci USA 89, (1992)

34 En RNA molekyl mer än en funktion Genombrottsmolekylen: hur skapades den? "One RNA two folding possibilities" Hur många förändringar? Virtanen and Kirsebom (1997) NFR årsbok Schultes and Bartel Science 289 (2000)

35 En RNA molekyl mer än en funktion Genombrottsmolekylen: hur skapades den? "One RNA two folding possibilities" Få!!!! Virtanen and Kirsebom (1997) NFR årsbok Schultes and Bartel Science 289 (2000)

36 Compartmentalization? Ribonucleotide reductase! Szostak et al. (2001), Nature 409, 387

37 Till slut så fanns det en RNA-värld! prebiotisk kemi, pre-rna värld Virtanen & Kirsebom, NFRs årsbok 1997 Tidsaspekten!

38 Framtiden Other functions Virulence Diagnostics Drug targets Regulators Host defence "RNA and disease" Tillämpning

39 "RNA and disease" Infectious diseases - A global threat Multi resistant bacteria e.g. MRSA Tuberculosis Globalization and spread Climate change Emerging diseases - Buruli ulcer Leptospirosis Virus infections - HIV, Influenza, SARS

40 Protein syntesen "RNA and disease"

41 "RNA and disease" New Findings New possibilities and strategies Antibiotics and RNA target should be unique and essential for the pathogen in bacteria there are many! Mikkelsen et al. (2001) Nature Struc. Biol. 8, Binding of paromomycin and Me 2+ overlap!

42 RNA-världen: forntid i nutid med framtid Thank you!