Evolution and the Genetic Code

1. Evolution and the Genetic Code RNA World to DNA Code Karp/CELL & MOLECULAR BIOLOGY 3E

2. Which came first - proteins or DNA? • Ribozymes: both enzyme and genome • RNA world? • Later, RNA's functions were taken by DNA & protein • RNA was left as a go-between in flow of genetic information • Splicing may be example of legacy from an ancient RNA world Karp/CELL & MOLECULAR BIOLOGY 3E

3. Which came first - proteins or DNA? • Group II introns found in purple bacteria & cyanobacteria • chloroplast-mitochondria ancestors • group II introns may be source of pre-mRNA introns • endosymbiotic organelles carried introns into eukaryotes • introns left organelle DNA & invaded nuclear DNA • this “exodus” occurs at high frequency Karp/CELL & MOLECULAR BIOLOGY 3E

4. Which came first - proteins or DNA? • Introns may have spread via transposition • some modern introns can still act like mobile genetic elements • self-splicing: excised themselves from 1° transcript • catalytic intron fragments copied to separate genome locations • "new" independent splicing genes • evolved into snRNAs: depend on proteins • snRNP become components of the spliceosome • Internal intron nucleotides lost function • hence, variable length & divergent sequences Karp/CELL & MOLECULAR BIOLOGY 3E

5. Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.39

6. Introns: both value and burden • RNA splicing is regulated • Alternative splicing: optional introns and exons • same gene, many proteins • snoRNAs encoded by introns not exons • within genes for ribosomal proteins, translation factors • introns excised, processed into snoRNAs • Several genes have introns & exons “reversed” • introns make snoRNAs, exons degraded (no mRNA) Karp/CELL & MOLECULAR BIOLOGY 3E

7. Introns: a major impact on biological evolution • Exon shuffling • Many proteins/genes are chimeric • Composites of parts of other genes • Reflects shuffling of genetic modules • Introns act as inert spacer molecules • Allows new sequence at junctions without affecting function Karp/CELL & MOLECULAR BIOLOGY 3E

8. Introns: a major impact on biological evolution • Easy recombination speeds evolution • Not limited to accumulation of point mutations • Allows “jump forward” evolution • Old parts in new context Karp/CELL & MOLECULAR BIOLOGY 3E

9. Ribozyme Update • To date, very few activities • Cleavage & ligation of phosphodiester bonds • Mostly RNA • Formation of peptide bonds during protein synthesis • Catalytic RNAs from “scratch” • Let automated DNA-synthesis of random DNA • Transcription of DNAs to RNA population • Select RNAs from population by activity • Molecular evolution in lab (In vitro evolution) Karp/CELL & MOLECULAR BIOLOGY 3E

10. Ribozyme Update • Selection via affinity chromatography • RNAs that bind ligand stick to column • Cycle between selection and mutation • Increase stringency of selection • Increase binding affinity for ligand • First step to catalysis: binding • Second round of selection for catalysis Karp/CELL & MOLECULAR BIOLOGY 3E

11. Ribozyme Update • Examples of ribozymes evolved de novo • ATP binding, then kinase (phosphorylation) • RNA polymerase • Aminoacyl-tRNA-synthetase (aa to RNA) Karp/CELL & MOLECULAR BIOLOGY 3E

12. Ribozyme Update • Selection via affinity chromatography • RNAs that bind ligand stick to column • Cycle between selection and mutation • Increase stringency of selection • Increase binding affinity for ligand • First step to catalysis: binding • Second round of selection for catalysis Karp/CELL & MOLECULAR BIOLOGY 3E

13. Ribozyme Update • RNA World • Amino acids perhaps only cofactors for ribozymes • Then, ribozymes to make peptides from amino acids • Then, RNA world became RNA-protein world • Later, RNA genome replaced by DNA • DNA evolution might require only 2 types of enzymes • ribonucleotide reductase (make DNA nucleotides) • reverse transcriptase (make DNA copies of RNA) • RNA catalysts not involved in DNA synthesis • RNA catalysts not involved in transcription • Supports idea that DNA was the last to appear • At some point, genetic code evolved Karp/CELL & MOLECULAR BIOLOGY 3E

14. Genetic Code • Discovery of mRNA led to decoding • George Gamow, physicist – proposed triplet code • 20 aa’s needed at least 3 letter code (64 of them) • Also proposed code was overlapping (wrong) • Code is degenerate • Most aminos coded for by >1 codon • 3 codons are termination codons Karp/CELL & MOLECULAR BIOLOGY 3E

15. Figure 11.40 Karp/CELL & MOLECULAR BIOLOGY 3E

16. Genetic Code • Marshall Nirenberg & Heinrich Matthaei • made artificial genetic messages • determined protein encoded in cell-free protein synthesis • Cell-free protein synthesis system • bacterial extract • 20 amino acids • poly(U) makes polyphenylalanine • di-nucleotide, tri-, tetra, etc. Karp/CELL & MOLECULAR BIOLOGY 3E

17. Genetic Code • Code essentially universal • exceptions (mostly in mitochondrial mRNAs) • human mitochondria • UGA is tryptophan not stop • AUA is methionine not isoleucine • AGA & AGG are stops not arginine Karp/CELL & MOLECULAR BIOLOGY 3E

18. Genetic Code • Codon assignments not random; • codons coding for same aa generally similar • mutations in one base often do not change aa • Similar amino acids coded for by similar codons • hydrophobic aa codons similar • conservative substitutions • third nucleotide most variable • glycine has 4 codons, all start with GG Karp/CELL & MOLECULAR BIOLOGY 3E

19. Figure 11.41 Karp/CELL & MOLECULAR BIOLOGY 3E

20. Codons and tRNA’s • Adaptor Molecule Proposed by Crick • tRNA’s discovered soon after • Robert Holley (Cornell, 1965) sequenced yeast alanine-tRNA • Small (73 – 93 bases) • Unusual bases altered posttranscriptionally • Secondary structure • Cloverleaf-like secondary structure (stems & loops) • Amino acid attaches to CAA at 3' end • Unusual bases mostly in loops Karp/CELL & MOLECULAR BIOLOGY 3E

21. Figure 11.42 Karp/CELL & MOLECULAR BIOLOGY 3E

22. Codons and tRNA’s • tRNAs tertiary structure • X-ray diffraction • 2 double helices arranged in shape of an L • invariant bases responsible for universal shape • must also have unique patterns to be charged correctly Karp/CELL & MOLECULAR BIOLOGY 3E

23. Codons and tRNA’s • Middle tRNA loop has anticodon • H bonds to mRNA codon • Loop has 7 bases (middle 3 anticodon) • opposite end of L has amino acid • third position of codon less important: wobble • 16 codons end in U: change to C gives same amino • third site A to G usually does not change amino acid Karp/CELL & MOLECULAR BIOLOGY 3E

24. Figure 11.43a Karp/CELL & MOLECULAR BIOLOGY 3E

25. Codons and tRNA’s • Rules for wobble • U of anticodon can pair with A or G of mRNA • G of anticodon can pair with U or C of mRNA • I (inosine, similar to guanine) pairs with U, C or A Karp/CELL & MOLECULAR BIOLOGY 3E

26. Figure 11.44 Karp/CELL & MOLECULAR BIOLOGY 3E

27. Matching tRNAs to aa’s • Amino acid activation • Performed by aminoacyl-tRNA sythetases (AAS) • Each amino acid recognized by specific AAS • AASs surprisingly different in sequence/structure • AASs “actuate” the genetic code • AASs carry out two-step reaction: • ATP + amino acid —> aminoacyl-AMP + PPi • aminoacyl-AMP + tRNA —> aminoacyl-tRNA + AMP Karp/CELL & MOLECULAR BIOLOGY 3E

28. Matching tRNAs to aa’s • AAS 3D structure determination by X-ray crystallography • find AAS sites that contact tRNAs • the acceptor stem & the anticodon most important • targeted mutagenesis • Find what makes tRNA charged by wrong AAS • alanyl-tRNA G-U base pair (3rd G from 5' end) • Insert G-U into acceptor stem of tRNAPhe or tRNACys • Causes alanyl-AAS to add alanine to these tRNA’s Karp/CELL & MOLECULAR BIOLOGY 3E

29. Matching tRNAs to aa’s • Notes on charging reaction • ATP energy makes aminoacyl-AMP • PPi hydrolyzed to Pi, further driving reaction forward • AAS has one of two proofreading mechanisms • Severs amino acid • Hydrolyzes AMP-aa bond • The leucyl-tRNA synthetase employs both types of proofreading • Valine & leucine differ by single methylene group Karp/CELL & MOLECULAR BIOLOGY 3E

30. Matching tRNAs to aa’s • AA itself plays no role • Fritz Lipmann et al. • Chemically altered amino acid after charged • Charged cysteine converted to alanine • Alanine inserted in place of cysteine Karp/CELL & MOLECULAR BIOLOGY 3E

31. Figure 11.46 Karp/CELL & MOLECULAR BIOLOGY 3E