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The Genetic Code and Transcription


No direct DNA participation in translation ... Cell-free translation system. In 1961 mRNAs not yet isolated. polynucleotide phosphorylase ... – PowerPoint PPT presentation

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Title: The Genetic Code and Transcription

Chapter 13
  • The Genetic Code and Transcription

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
  • translation

Information Flow in Cells
  • Transcription
  • Translation
  • Genetic code

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

Early Studies
  • 1950s
  • became clear mRNA serves as intermediate in
    transferring genetic information from nucleus to
  • 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

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
  • 3 (not 2) seemed to be the key multiple
  • Triplet code

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

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
  • 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
  • Crick
  • Overlapping code unlikely due to physical
    constraints during translation (and predicts
    adapter by hydrogen bondingtRNAs)

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

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

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
  • Controlled mixtures
  • High A, low C give predictable nucleotides (AAA,
    AAC, ACA and CAA)

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

Translation of Ribonucleotide Homopolymers In
Working Out the Codons
  • Translate homopolymers
  • Translate mixed (2 nucleotide) polymers
  • calculate theoretical codon frequency
  • Determine amino acid frequency in peptides

Copolymer Experiment
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

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

Genetic Code Summary
  • 64 codons
  • 61 call for amino acids (1 to 6 each)
  • degenerate
  • 3 triplets are stop signals
  • AUG is for methionine and also is the initiation

The Code
  • In the format suggested by Crick

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

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

An Ordered Code
  • Codons for a particular amino acid generally
  • 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

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

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

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

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

  • (b) shows relative positions of 7 fX174 genes

  • 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

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

Elliot Volkins Results
Brenner, Jacob and Meselson
  • Are individual ribosomes specific for a single
  • 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
  • Consistent with a DNA-derived mRNA being
    translated by a generic ribosome
  • Jacob and Monod model proposed in 1961

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
  • 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

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

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

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

Prokaryotic Transcription
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
  • mRNAs are monocistronic in eukaryotes

Transcription in Eukaryotes
  • Transcription in the nucleus, translation in
  • 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

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
  • Involves splicing out of intron-derived sequences
    (vs. exons) from transcript

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

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

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

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

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

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
  • Terminator causes clamp to open and complex
  • Structure conserved to human enzyme and 9 of 10
    subunits found conserved in RNAP I and RNAP III

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

Heteroduplex With Loop Outs
Genes With Introns
Genes and mRNA Sizes
RNA Splicing
  • Commonly involves ribozymes
  • snRNAs, snRNPs
  • Some RNAs are self-splicing
  • Tetrahymena rRNA transcripts, group I introns
  • Thomas Cseh, 1982

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

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
  • 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)

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

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

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
  • rDNA in amphibian oocytes
  • Drug resistance in eukaryotic cell cultures
  • Also occurs in some/many cancers
  • Prostate cancer? DMs and HSRs
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