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Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Evolution and the Genetic Code RNA World to DNA Code

Evolution and the Genetic Code

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Evolution and the Genetic Code. RNA World to DNA Code. 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 - PowerPoint PPT Presentation

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Page 1: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Evolution and the Genetic Code

RNA World to DNA Code

Page 2: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 3: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 4: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 5: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.39

Page 6: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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)

Page 7: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 8: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 9: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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)

Page 10: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 11: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Ribozyme Update

• Examples of ribozymes evolved de novo

– ATP binding, then kinase (phosphorylation)

– RNA polymerase

– Aminoacyl-tRNA-synthetase (aa to RNA)

Page 12: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 13: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 14: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 15: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.40

Page 16: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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.

Page 17: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 18: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 19: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.41

Page 20: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 21: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.42

Page 22: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 23: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 24: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.43a

Page 25: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 26: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.44

Page 27: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 28: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 29: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 30: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

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

Page 31: Evolution and the Genetic Code

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.46