Download Evolution and the Genetic Code

Evolution and the Genetic
Code
RNA World to DNA 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
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.39
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)
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
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
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)
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
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)
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
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.40
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.
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.41
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.42
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.43a
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.44
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
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
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.46