The Genetic Code and Transcription

1
Chapter 13

• The Genetic Code and Transcription

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Flow of Genetic Information in Cells

• Central dogma of molecular biology
• DNA replicates
• replication
• RNA is transcribed from a DNA template
• transcription
• mRNA templates are translated into proteins by
  ribosomes
• translation

3
Information Flow in Cells

• Transcription
• Translation
• Genetic code

4
Genetic Code

• Linear order of ribonucleotide bases derived from
  complementary DNA
• Codons call for amino acids and are triplets of
  nucleotides
• Codons are unambiguous and nonoverlapping
• Degenerate
• Code includes initiation and termination codons
• No internal punctuation
• Code is universal (with minor exceptions)

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Early Studies

• 1950s
• became clear mRNA serves as intermediate in
  transferring genetic information from nucleus to
  cytoplasm
• No direct DNA participation in translation
• Code thought to be overlapping to allow 4
  nucleotides call for 20 amino acids
• 1961
• Jacob and Monod postulated existence of mRNAs

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Triplet Code

• Sidney Brenner (early 1960s)
• Postulated triplet code based upon theoretical
  grounds (41 4, 42 16, 43 64)
• Crick, Barnett, Brenner and Watts-Tobin
• Deletion/insertion mutants in T4 rII locus
• Caused all subsequent amino acids to be wrong
• Reversions by subsequent mutations involved one
  insertion and one deletion, 3 insertions or 3
  deletions
• 3 (not 2) seemed to be the key multiple
• Triplet code

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Triplet Code

• Frameshifts by single deletions or insertions
• Single nucleotide insertions compensate for
  single nucleotide deletions
• Two single nucleotide insertions still give
  frameshift
• A total of 3 added or deleted nucleotides leave
  code in frame

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Nonoverlapping Code

• Brenner (early 1960s)
• Theoretical considerations make overlapping code
  highly unlikely
• Only certain amino acids could follow other
  certain amino acids since the first two
  nucleotides of the second codon would be already
  determined
• But looking at known amino acid sequences of
  proteins available at the time this was clearly
  not true
• Effects of single nucleotide insertions/deletions
  argue against overlapping code
• Would not affect all subsequent amino acids in
  protein
• Crick
• Overlapping code unlikely due to physical
  constraints during translation (and predicts
  adapter by hydrogen bondingtRNAs)

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More from Francis Crick

• Adapter hypothesis
• Predicted no internal punctuation on basis of
  genetic data available
• Only 20 of 64 possible codons specify amino acids
• Cant always be correct
• Insertion/deletion data suggested that all/most
  codons could be translated so he changed his
  opinion

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Code is Degenerate

• Degenerate
• Amino acids may be encoded by more than one codon
• Amino acids have up to 6 different codons

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Marshall Nirenberg and Matthaei

• 1961
• Cell-free translation system
• In 1961 mRNAs not yet isolated
• polynucleotide phosphorylase
• Can make synthetic ribonucleotide chains
• Normally degrades mRNAs but in high rNDP
  concentrations reaction runs in reverse
• Can make homopolymers
• AAAAAn, UUUUUn, CCCCCn, GGGGGn
• Controlled mixtures
• High A, low C give predictable nucleotides (AAA,
  AAC, ACA and CAA)

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Nucleotide Phosphorylase

• Can synthesize ribonucleotide polymers from rNDPs
• Normally degrades mRNAs by phosphorolysis in the
  cell

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Translation of Ribonucleotide Homopolymers In
Vitro
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Working Out the Codons

• Translate homopolymers
• Translate mixed (2 nucleotide) polymers
• calculate theoretical codon frequency
• Determine amino acid frequency in peptides

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Copolymer Experiment
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Triplet Binding Assays

• Nirenberg and Philip Leder
• 1964
• Synthesize trinucleotides of known sequence
• Ribosomes can bind mRNA as short at 3 nucleotides
  under proper conditions
• Form complex with tRNA
• Codon of mRNA binds to anticodon of tRNA
• This approach by several laboratories eventually
  assigns 50 of 64codons

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Triplet Binding Assay
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Translation of Repeating Copolymers

• Gobind Khorana, 1960s
• Synthesized long RNAs with dinucleotide,
  trinucleotide or tetranucleotide repeats
• For (UG)n there are only two possible triplets
• UGU and GUG ? cysteine and valine in peptide

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Results of Synthetic Copolymer Experiments

• (GAUA)n and (GUAA)n experiments suggested some
  codons do not translate to amino acids

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Genetic Code Summary

• 64 codons
• 61 call for amino acids (1 to 6 each)
• degenerate
• 3 triplets are stop signals
• UAA, UAG, UGA
• AUG is for methionine and also is the initiation
  codon

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The Code

• In the format suggested by Crick

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More Crick Hypotheses

• Organized codons into now accepted chart format
• Noticed nearly all degeneracy was in the 3rd
  position of the codon
• Proposed wobble hypothesis in 1966
• First two nucleotides more critical
• Base pairing by 3rd nucleotide of codon (to
  anticodon of tRNA) could be less constrained
• Wobble pairing

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Wobble Pairing

• 1st position of anticodon with 3rd position of
  codon
• 1st position of anticodon allowed to pair with up
  to 3 different bases in 3rd position of codon
• Inosine base

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An Ordered Code

• Codons for a particular amino acid generally
  grouped
• Amino acids with similar properties (hydrophobic,
  positive charge, etc.) often have at least 2
  position nucleotide of codon in common
• Buffers the effect of mutations

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Initiation

• Nearly all protein coding sequences begin with
  AUG
• Initiator codon
• Rarely GUG
• Recognized by special initiator tRNA carrying
  methionine
• All polypeptides begin with methionine
• N-formylmethionine in prokaryotes

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Termination

• 3 termination codons
• UAA, UAG, UGA
• Often called nonsense codons
• Amber, ochre, umber
• Not recognized by normal tRNAs
• Recognized by special proteins called releasing
  factors
• Mutations in tRNA gene in anticodon region can
  produce suppressor tRNAs that suppress stop
  signals (and therefore nonsense mutations

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Confirming the Code

• 1972, Walter Fiers
• MS2 bacteriophage
• RNA chromosome, 3 genes, 3500 nucleotides
• Compared amino acid sequence of coat protein and
  the gene (RNA) that encoded it
• Agreed with predicted translation
• AUG start, UAAUAG double stop codons
• Note RNA sequence compared, DNA could not yet be
  sequenced

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Nearly Universal

• Up to 1978 considered universal
• Humans and E. coli use same basic code, as do all
  other species
• 1979 noted that coding properties of human and
  yeast mtDNA genes not quite the same as predicted
• In general exceptions simplify the code
• Reduce number of tRNAs required for translation
  in mitochondria (only 22 encoded there)

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Overlapping Genes

• Genetic code is nonoverlapping
• But genes sometimes overlap
• Generally for only a short distance because of
  constraints placed on both peptide sequences
• Mostly in viruses, some bacterial genes
• Optimizes the use of DNA coding space

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• (b) shows relative positions of 7 fX174 genes

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Transcription

• Synthesis of RNA from a DNA template
• Suggestive observations that RNA is an
  informational intermediate between DNA and the
  site of protein synthesis
• DNA in chromosomes in nucleus, proteins made by
  ribosomes in cytoplasm
• RNA synthesized in nucleus and is chemically
  similar to DNA
• After synthesis, much RNA migrates to cytoplasm
• Amount of RNA in cell is generally reflective of
  the level/amount of protein synthesis

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Evidence for mRNAs

• Volkin, 1956 and 1958
• 32P, T2 and T7 phage, E. coli
• Added 32P to culture medium as cells were
  infected with bacteriophage
• Newly made (radioactive) RNA matched composition
  of phage DNA, not original E. coli RNA

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Elliot Volkins Results
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Brenner, Jacob and Meselson

• Are individual ribosomes specific for a single
  protein?
• Heavy isotope-labeled E. coli ribosomes, 1961
• Determined that preinfection ribosomes
  synthesized proteins from phage genes not present
  in the cell when the ribosomes were themselves
  synthesized
• Consistent with a DNA-derived mRNA being
  translated by a generic ribosome
• Jacob and Monod model proposed in 1961

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RNA Polymerase

• Discovered in 1959
• n(rNTP) DNA RNAP ? (rNMP)n n(PPi)
• Nucleotides linked by 5 to 3 phosphodiester
  linkages and made 5?3
• Pyrophosphate subsequently cleaved by
  pyrophosphatase
• E. coli holoenzyme composed of a2bbs
• Core enzyme (a2bb) synthesizes RNA while s
  recognizes promoter sequence
• E. coli has only one type of RNA polymerase

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E. coli Promoters

• Initial step of RNA synthesis is template binding
  by sigma factor at the promoter
• Promoters are transcription start regions
• Sigma binds to 60 bp of DNA about 40 nucleotides
  upstream and 20 downstream from the actual
  transcription start point
• Promoters can be strong or weak
• Start frequency of every 1-2 seconds, up to once
  per 20-30 minutes

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Consensus Sequences

• Conserved sequences found 10 and 35 nucleotides
  upstream of the transcription start point (1)
• Called 10 (Pribnow box, TATA box) and 35
  sequences (TTGACA)
• Cis-acting elements
• Bound by trans-acting factors
• Common E. coli s factor is s70
• Others are s32 s54 sS sE and have different 10
  and -35 sequences

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Phases of Transcription

• Initiation
• No primer required, sigma is
• Short 8-mer oligonucleotide synthesized
• Can be abortive
• Elongation
• Sigma lost, holoenzyme, 5 to 3
• 50 nucleotides/sec at 37 degrees Celsius
• Termination
• Hairpin structure in RNA
• Rho-dependent or independent

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Prokaryotic Transcription
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mRNA Molecules

• Can be polycistronic in prokaryotes
• Operons lead to mRNAs with multiple genes
• mRNAs can associate with ribosomes and begin
  translation before transcription is completed
• Transcription and translation are said to be
  coupled
• mRNAs are monocistronic in eukaryotes

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Transcription in Eukaryotes

• Transcription in the nucleus, translation in
  cytoplasm
• Chromatin must be uncoiled and DNA made
  accessible to RNA polymerase
• Chromatin remodeling
• Initiation involves a more complex set of
  interactions between cis-acting elements and
  trans-acting factors
• Initiation factors, enhancers
• Elements can be within or downstream from gene

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Eukaryotic Pre-mRNA Processing

• Addition of CAP to 5end
• polyA tail to most 3 ends
• Initial RNA molecules called primary transcripts
  or hnRNAs (that form hnRNPs)
• Perhaps as few as 25 of hnRNAs converted to
  mRNAs
• Involves splicing out of intron-derived sequences
  (vs. exons) from transcript

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Eukaryotic RNA Polymerases

• Eukaryotes have 3 different RNA polymerases
• Each specialized for production of particular
  types of RNA

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Transcriptional Initiation in Eukaryotes

• RNAP II transcribes pre-mRNAs (hnRNAs)
• In yeast has 12 subunits/polypeptides
• Regulated by transacting factors and
  core-promoter, promoter (includes elements in
  addition to the core promoter element) and
  enhancer elements
• Core promoter element is called the
  Goldberg-Hogness or TATA box
• Common consensus is TATAAAA
• Similar to E. coli 10 but located 25 to -30

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Other Promoter Elements

• CAAT box
• Consensus GGCCAATCT
• Generally upstream of TATA and commonly within
  100 bp
• Distance and orientation may vary
• Basal element
• GC box
• Properties generally similar to CAAT
• Enhancer element
• Act at great distances upstream and downstream
• Associated with very strong promoters

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Transcription Factors

• Factors are proteins
• Generalized transcription factors
• Required for all RNAP II mediated transcription
• Bind to basal elements
• Required for polymerase binding, do not turn
  gene on/off
• Specific transcription factors
• Involved in regulating on/off, specific for gene
  or subset of genes

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RNAP II Transcription Factors

• Designated TFIIA, TFIIB, TFFIIetc
• Complex, TFIID has 10 polypeptides
• One is TBP or TATA-binding protein
• Once binds at least 7 other general transcription
  factors bind to form pre-initiation complex,
  which then binds RNAP II

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Eukaryotic Transcription

• Yeast modelRoger Kornberg (Arthurs son)
• Two large subunits, 10 others, 500kDa
• Has positive-charged cleft to bind DNA, which
  clamps around DNA
• Initial interaction/synthesis is unstable and
  process often aborts by 11-mer
• If proceeds beyond this point will continue until
  termination
• Terminator causes clamp to open and complex
  dissociates
• Structure conserved to human enzyme and 9 of 10
  subunits found conserved in RNAP I and RNAP III

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Eukaryotic RNA Processing

• hnRNA is converted to mRNA
• Posttranscriptional modifications
• 7-methyl guanosine nucleotide cap attached 5 to
  5 to the 5 end of transcript, 2 of terminal
  sugar(s) also methylated
• May be essential for transport out of the nucleus
  and protect from 5 exonuclease attack
• PolyA added to 3 end
• About 200-250 nucleotides by polyA polymerase
• Signal is AAUAAA (actually for nuclease cleavage
  to produce mature 3 end of hnRNA
• Without polyA transcript is degraded

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Intervening Sequences in Eukaryotic Genes

• Intervening sequences, split genes
• Introns
• Exons
• Discovered when genomic DNA hybridized to mRNAs
  or cDNAs
• Heteroduplex had loop outs
• Common to most genes
• More (can be 50) and larger in higher eukaryotes
  (genes can be 5X or more than mRNA, dystrophin
  mRNA is 1 of gene)
• Locations conserved, sizes/sequences not

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Heteroduplex With Loop Outs
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Genes With Introns
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Genes and mRNA Sizes
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RNA Splicing

• Commonly involves ribozymes
• snRNAs, snRNPs
• Some RNAs are self-splicing
• Tetrahymena rRNA transcripts, group I introns
• Thomas Cseh, 1982

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Group I Introns

• Self-splicing
• Catalytic activity in the intron itself
• Requires a guanosine nucleoside or nucleotide for
  hydroxyl group
• Involves two nucleophylic attacks,
  transesterifications
• Found in ciliate rRNA transcripts and some
  organelle mRNA and tRNA primary transcripts

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Splicesomes and Nuclear Splicing

• Nuclear introns can be up to 20 K nucleotides
• Nuclear introns commonly begin (5, donor
  sequence) with GU and end with AG (3, acceptor
  sequence)
• Splicing carried out by large spliceosome complex
  (40S in yeast, 60s in mammals)
• Small nuclear RNAs (snRNAs)/small nuclear
  ribonucleoproteins (snRNPs or snurps)
• U1, U2, U3, U4, U5, U6 (rich in Uridine bases)

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Spliceosome Mechanism

• U1 complementary to 5 site
• Two transesterifications
• Hydroxyl comes from adenylate residue at branch
  site bound by U2
• Branch site attacks 5 end of intron
• Free end of exon attacks 3 end of intron,
  releasing intron as a lariat structure

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RNA Editing

• Change in the nucleotide sequence of a pre-mRNA
  prior to translation
• Actual final sequence not found in DNA
• Can be substitution editing (change base) or
  insertion/deletion editing
• Classic example Trypanosoma genus
• Some RNAs (e.g. cox) have 60 of their total
  transcript added after transcription completed
• Uridines
• gRNAs (guide RNAs) provide complementary template
• Mammalian example
• Long and short forms of apolipoprotein B
• CAA to UAA

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Transcription and Translation

• Coupled in prokaryotes
• Not coupled in eukaryotes
• Visualized by electron microscopy

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Gene Amplification

• Temporary increase in the copy number of certain
  genes
• rDNA in amphibian oocytes
• Drug resistance in eukaryotic cell cultures
• Also occurs in some/many cancers
• Prostate cancer? DMs and HSRs