Chapter 18: Outline

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Chapter 18 Outline

• Genetic Information
• DNA Replication
• DNA Repair
• DNA Recombination
• Transcription
• Prokaryotes
• Eukaryotes
• Gene Expression
• Prokaryotes
• Eukaryotes

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18.1 Genetic Information

• All living organisms must be able to
• Rapidly and accurately synthesize DNA
• Repair DNA to ensure genetic stability
• We will focus on the above goals, primarily in
  prokaryotes since the process is better
  understood with them.

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Information Flow

• DNA RNA Protein

Translation
Transcription
Replication
Translation Protein is synthesized from AAs and
the three RNAs.
4
DNA Replication General

• DNA can be double helix, single strand
• linear, circular
• To duplicate a double helix
• 1. Separate strands while protecting
• single strands formed.
• 2. Synthesize the DNA always from the
• 5 to 3 end.
• 3. Protect against any errors in the
• replication process.

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Semiconservative Replication

• Each separated DNA strand is duplicated to give
  two new double helices.
• Meselson and Stahl used 15N-labeled DNA as a
  starting point. New DNA was synthesized using
  normal 14N DNA nucleotides as feedstock.
• Density-gradient centrifugation showed only
  14N-15N new DNA after one generation and a 50-50
  mix of 14N and 14N-15N after two generations.

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Semiconservative Replication2

• Diagram shows the N distribution.

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DNA Synthesis, Prokaryotes

• DNA unwinding via helicase (Dna B in E. Coli.)
• Primer synthesis of short RNA segments catalyzed
  by primase, an RNA polymerase. The primosome
  contains primase and several auxiliary proteins.
• DNA synthesis using a template strand to form
  phosphodiester bonds between nucleotides
  catalyzed by DNA polymerase. Pol III is major.

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DNA Synthesis-2, Nucleophilic Attack
5
Bases 1 and 2 must be com- plimentary to bases in
template chain of DNA.
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DNA Synthesis-3

• DNA polymerase III (pol III) is the major
  synthesis enzyme in prokaryotes and has at least
  10 subunits.
• Core a, e, and K subunits
• The t subunit joins two core units to form a
  dimer.
• b-protein (sliding clamp) of two units forms a
  ring around the template strand.

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DNA Synthesis-3 (cont.)

• g complex recognizes single strands with primer
  and transfers the b-protein to the core
  polymerase and prevents frequent dissociation
  (processivity).
• The DNA replicating machine is called the
  replisome.
• Pol I is a repair enzyme.
• Pol II is not well understood.
• All three Pols are 3?5 exonucleases.

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DNA Synthesis-3 (cont. sub units Pol III)
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DNA Synthesis, Prokaryotes (cont.)

• 4. Joining DNA fragments occurs via DNA ligase.
• 5. Supercoiling control is via DNA topoisomerases
  which function ahead of the replication machinery
  to relieve torque.
• Type one topoisomerases cause transient
  single-strand breaks in DNA type two cause
  transient double-strand breaks in DNA.

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Supercoiling, cont.

• DNA gyrase (prokaryotes) helps separate
  replication products and creates negative
  supercoils required for genome packaging.

14
Bidirectional Replication

• The helix begins to unwind at the origin of
  replication (Ori).
• At each Ori are two replication forks with the
  new DNA chains being synthesized at each fork.
• Prokaryotes have one Ori in a supercoiled helix,
  eukaryotes have multiple Ori.

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DNA Replication
The DNA duplex at the replication fork is unwound
by helicase. DNA gyrase introduces negative
supercoils ahead of rep fork. Lagging strand is
looped to allow simultaneous synthesis of both
new strands.
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DNA Replication

• Primase copies a short stretch of DNA as an RNA
  primer at the 5 end and serves as an anchor
  point to begin synthesis of DNA.
• Replisome is a multiprotein complex

Primosome
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DNA Replication

• Pol I removes primer and replaces it with
  deoxynucleotides.
• DNA ligase seals the nicks on the polymer and
  joins the Okazaki fragments.

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DNA Replication

• The most common post synthesis modification of
  DNA is methylation.
• In prokaryotes, adenine and thymine are the bases
  usually metylated. In eukaryotes, cytosine is
  methylated.
• Methylation serves to protect the DNA of bacteria
  from restriction endonucleases while allowing
  these nucleases to cut and destroy the DNA of
  invading bacteriophages.

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DNA Replication Eukaryotes

• Timing is limited to a specific period referred
  to as the S phase.
• Replication rate is slower, approx-imately 50
  nucleotides/sec per rep fork.
• Multiple replicons speed overall replication to
  just a few hours.
• Okazaki fragments are 100-200 nucleotides long.
  (Much shorter)

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DNA Replication Eukaryotes-2

• Polymerases a, b, d, e, and g are found in
  eukaryotes.
• Pol a- e are found in the nucleus and g in the
  mitochondria.
• The a enzyme is responsible for the initiation of
  synthesis and d for continuation of the chain.
• Replication protein A (RPA) prevents reforming of
  the helix.
• FEN1 removes primers.

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DNA Replication Eukaryotes-3

• A DNA ligase again seals the nicks.
• DNA Pol d binds to proliferating cell nuclear
  antigen (PCNA), a sliding clamp (like b in E.
  coli.).
• Histones must be synthesized concurrently with
  the DNA.

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DNA Repair

• Without removal of nucleotides
• Broken phosphodiester bonds repaired by DNA
  ligase.
• Pyrimidine dimers restored by photo-reactivation
  repair by DNA photolyase. Many species (e. g.
  humans) do not have the enzyme.

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DNA Repair

• With removal of nucleotides
• In excision repair, mutations are excised by
  enzymes that remove incorrect bases and replace
  them with the correct ones.
• An exinuclease removes 12-13 bases (27-29 in
  eukaryotes).
• In E. coli the exinuclease is composed of the
  proteins UvrA, UvrB, and UvrC. See the next
  slide.

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DNA Repair-3
Fig 18.12
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DNA Repair

• Recombinational repair occurs when replication is
  interrupted and a gap occurs opposite a damaged
  site (e. g. pyrimidine dimers).
• The gap is repaired by exchanging the
  corresponding segment of the homologous DNA
  molecule. The gap in the homologeous molecule
  is repaired by DNA polymerase and ligase.

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DNA Recombination

• The two forms are
• General which occurs between homologous DNA
  molecules.
• Site-specific in which the exchange of sequences
  from different molecules requires only short
  sequences of DNA homology.
• Transposition is a variant of site-specific
  recombination in which transposable elements are
  moved from one chromosome or region to another.

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General Recombination

• Two homologous DNA molecules pair.
• One strand in each molecule is cleaved.
• The strands cross over making a Holliday
  intermediate.
• DNA ligase seals cut ends.
• Branch migration leads to transfer of DNA between
  homologs.
• A second series of strand cuts occurs.
• DNA polymerase fills any gaps and DNA ligase
  seals the strand cuts.

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General Recombination, E. coli

• Initiated by RecBCD (exonuclease and helicase)
  which cleaves strands until reaching
  5-GCTG-GTGG-3 (Chi site).
• Strand exchange is effected by RecA which helps
  to form a triple helix.
• Branch migration occurs as RecA binds to the
  Holliday junction.
• Branch migration is catalyzed by RuvAB.
• Migration ends at 5-(AorT)TT(GorC)-3
• RuvC cleaves crossover strands and Holliday
  junction resolves.

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General Recomb. Gene Transfer

• In transformation, naked DNA breaches the cell
  wall and is incorporated in the genome.
• Transduction occurs when a bacteriophage
  inadvertently carries DNA to a recipient cell.
• With conjugation, a donor bacterial cell forms a
  sex pilus which attaches to the surface of the
  recipient cell. A fragment of the donor DNA is
  transferred via the pilus.

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Site-specific Recombination

• Depends more on protein-DNA interactions.

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Transposition

• McClintock (1940s) reported certain genome
  segments can move from one place to another.
• Transposable elements (transposons or jumping
  genes) were discovered in E. coli in 1967.
• Two mechanisms have been observed.

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Replicative Transposition

• A replicated copy is inserted in the new
  location.
• An intermediate called a cointegrate forms.
• Resolvase catalyzes a site-specific recombination
  allowing resolution of the cointegrate in to two
  separate molecules.

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Nonreplicative Transposition

• In nonreplicative transposition the transposable
  element is spliced out of the original site and
  inserted in the target site. The donor site must
  be repaired.
• With either form of transposition, short
  duplications of target site DNA are generated by
  the staggered cleavage catalyzed by transposase.

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Eukaryotic Transposons

• Some resemble those found in bacteria.
• Many eukaryotic transposons have different
  structures. E. g., the Ty transposon in yeast
  has long terminal repeats, involves an RNA
  intermediate, and bears a resemblance to the
  replicative phase of the retrovirus.

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Information Flow

• DNA RNA Protein

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18.2 Transcription

• The reaction catalyzed by all RNA polymerases is
• NTP (NMP)n ? (NMP)n1 PPi
• The direction of the gene is the same as the
  direction of the coding (-) strand of DNA.
• 5-TTTGGACAACGTCCAGC-3 () DNA
• 3-AAACCTGTTGCAGGTCG-5 (-)
• 5-UUUGGACAACGUCCAGC-3 RNA

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Transcription in Prokaryotes

• RNA polymerase from E. coli
• A multisubunit structure of the form a2bbws
• The holoenzyme loses the s subunit to give the
  core enzyme.
• The function of the s unit is to recognize the
  promoter locus.

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Sigma Units in E. coli.

• s70 (70 kD) promotes transcription of most genes.
• s32 promotes transcription of heat shock genes.
• s32 promotes transcription of the flagellin gene.

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Initiation Promoter Locus

• Promoter locus A DNA segment that signals the
  start of RNA synthesis.
• Is upstream (toward the 3 end) of the DNA
  segment where the gene coding for the RNA
  actually begins.
• Prokaryote promoter regions contain many
  sequences in common called consensus sequences.
  These are rich in A-T base pairs (two H bonds)

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Initiation Consensus Sequences

• In prokaryotes
• often 25 and 10 base pairs upstream of the start
  of transcription
• -35 box and 10 (Pribnow) box
• TTGACA TATAAT
• In eukaryotes
• TATA box lies 25 base pairs upstream

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Elongation

• The s unit leaves after about 10 nucleotides have
  added to the RNA.
• Core polymerase binds accessory proteins and RNA
  synthesis continues in the 5-3 direction.
• The transcription bubble (DNA-RNA hybrid)
  leaves positive supercoils ahead of and negative
  supercoils behind on the DNA as it moves
  downstream.

42
Termination (Prokaryotes)

• Termination sequences contain palindromes. The
  RNA transcript forms a hairpin turn which
  disrupts the DNA-RNA hybrid.
• In r-dependent terminmation, the r protein binds
  to RNA and catlayzes dissociation of the
  poylmerase from the DNA-RNA hybrid.
• mRNA is used immediately.
• Mature rRNA and tRNA are processed from larger
  transcripts. tRNA bases are frequently modified
  after transcription.

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Modification of RNAs

• 1. Leader and trailer sequences are removed.
  (Trimming)
• 2. Terminal sequences can be added.
• 3. Base modification can occur.

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Modification of RNAs tRNA

• Base modification occurs both before and after
  trimming. Methylation and substitution are
  common.
• Often several tRNAs are synthesized in one long
  chain which is split into fragments after
  synthesis.
• RNase P makes 5 ends of all E. coli tRNAs. The
  enzyme consists of both protein and RNA with the
  RNA being catalytically active.

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Modification of RNAs tRNA

• All tRNAs have a CCA sequence at the 3 end.
  This end is where the amino acid to be
  transferred to the ribosome is attached.
• tRNAs have a three-dimensional L shape

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Modification of RNAs rRNA

• rRNA processing consists mainly of methylation
  and trimming to proper size.
• There are three rRNAs in a prokaryotic ribosome
  and five in eukaryotic ribosomes. See the
  section on nucleic acid structure for a review of
  ribosome substructure.

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Modification of RNAs rRNA-2

• Individual Rnases are identified by
• letters/numbers, e. g. M5, X and III.

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Modification of RNAs mRNA

• Prokaryotic mRNA is used as synthesized.
• Eukaryotic mRNA is processed extensively
• 1. Capping occurs at the 5 end.
• 2. Polyadenylation occurs at the 3 end.
• 3. Splicing of code sequences (called exons)
  occurs and noncoding sequences (introns) are
  removed.

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Transcription (Eukaryotes)

• The three RNA polymerases differ in the type of
  RNA synthesized, subunit structure, and relative
  amounts.
• RNA Pol I transcribes large rRNA.
• RNA Pol II transcribes precursors to mRNA and
  most snRNAs.
• RNA Pol III transcribes precursors of tRNAs and
  5S rRNA.

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Transcription (Eukaryotes)

• 2. Promoters are larger and more complex.
• Often the TATA box 25-30 bp upstream promotes
  transcription by Pol II.
• Various complexes form, the DNA strands unwind at
  the TATA region, and the process begins.

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Transcription (Eukaryotes)-3

• 3. Processing is more complex for eukaryotic
  mRNA. hnRNA is associated with nuclear proteins
  in ribonuclearprotein particles (hnRNP).
• Cap structure protects 5 end from exo-nucleases.
  (Next slide)
• The 3 end is chopped and 100-250 adenylate
  residues are added to protect the end and promote
  export of mRNAs.

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Eukaryotic 5 cap (mRNA)
53
Exons, Introns, and Splicing

• Eukaryotic genes often contain base sequences
  that do not appear in the mRNA expressed by the
  gene. These sequences are called introns. They
  are cut from the RNA nucleotide chain and the
  exons (the bases that are expressed) are spliced
  together.
• The number of introns in a gene can vary from
  none to many (gt50).

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Exons, Introns, and Splicing

• Introns are removed in the nucleus by small
  nuclear ribonuclearproteins, snRNPs. (pronounced
  snurps)
• snRNPs function by cutting the mRNA, forming a
  lariat (loop) of the introns, and joining the two
  pieces of exon as the loop is cleaved. See the
  next slide.
• Some RNAs catalyze their own self-splicing.
• Lupus results from antibodies to a snRNP.

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Exons, Introns, and Splicing

• Splicing begins with nucleophillic attack of the
  2-OH on A on the 5 splice site.
• A 2-5 phospho-diester bond forms the lariat.
• The 3-OH attacks the phosphate next to the
  lariat.

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Ribozymes

• RNAs with catalytic activity are called
  ribozymes. The first ribozymes discovered
  catalyzed their own splicing.
• Group I ribozymes require a guanosine which is
  covalently bonded at the splice site.
• Group II ribozymes use a lariat mechanism and do
  not require a guanosine.

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18.3 Gene Expression (Intro)

• Regulation of gene transcription is a result of a
  complex hierarchy of control elements.
• Constituitive genes are routinely transcribed as
  their products are required for function.
• Inducible genes are expressed only under certain
  circumstances, e. g. the ones for lactose
  metabolism in E. coli.

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Gene Expression (Intro)-2

• Most mechanisms use the interactions between the
  base pairs in the major groove and proteins.
• 20 or more contacts involving hydrogen bonding,
  hydrophobic interactions, and ionic bonds result
  in highly specific DNA-protein interactions.
• Helix-turn-helix, helix-loop-helix, leucine
  zipper, and zinc finger motifs of proteins bind
  to DNA. (Next slide.)

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Gene Expression (Intro)-3
Gene regulatory proteins
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Gene Expression (Prokaryotes)

• Operons are groups of linked structural and
  regulatory genes.
• The lac operon (below) controls lactose
  metabolism.

Fig 18.28
61
Jacob and Monod Theory

• The inducible protein (b-galactosidase) is coded
  by a structural gene (Z).
• A regulatory gene (i) produces a repressor
  protein (RP) which binds at a site called the
  operon (O) and thereby inhibits synthesis of
  b-galactosidase because the promotor site is
  blocked to RNA polymerase.
• Next slide.

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• Repressor protein binds to operator site and
  prevents RNA polymerase from initiating synthesis.

Allolactose inactivates the repressor protein.
63
Gene Expression (Eukaryotes)

• Changes in amount and activities of gene products
  regulated by
• Genomic control
• Transcriptional control
• RNA processing
• RNA transport
• Translational control

64
Gene Expression (Eukaryotes)

• Genomic control may involve methylation and
  histone acetylation.
• Histone acetylation generally promotes gene
  expression.
• Gene rearrangements and gene amplification are
  less common examples of genomic control.

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Gene Expression (Eukaryotes)

• RNA processing takes a variety of forms.
• Alternative splicing-the joining of different
  combinations of exons-results in different mRNAs.
• Different sites for polyadenylation can affect
  mRNA function.
• With RNA editing, bases are chemically modified,
  deleted, or added. E. g. the C of a CAA codon
  may be converted to a UAA and a shorter protein
  results.

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Gene Expression (Eukaryotes)

• RNA transport through nuclear pore complexes is
  regulated by, for example, capping and by binding
  of the 5 end of mRNA within hnRNP to cap-binding
  protein.
• Covalent modification of several translational
  factors (nonribosomal proteins) enhances the
  translation of specific mRNAs.

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Signal Transduction

• In most cases changes in gene expression are
  initiated by the binding of a ligand to either a
  cell surface receptor or an intercellular
  receptor.
• Cell division is the most studied example of
  signal transduction because faulty cell division
  results in cancer.

68
Signal Transduction

• Complicating features of intracellular signal
  transduction include
• 1. Each type of transduction signal may activate
  one or more pathways.
• 2. Signal transduction pathways may converge or
  diverge.

69
Signal Transduction

• Progression through the four phases of cell
  growth (M, G1, S, and G2) is regulated by
  alternating synthesis and degradation of a group
  of proteins called the cyclins, a group of
  regulatory proteins that bind to and activate the
  cyclin-dependent protein kinases (Cdks).
• Cdks are a class of kinases that trigger passage
  of a cell through a checkpoint to the next phases
  of mitosis.

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Signal Transduction

• Positive control of cell growth is exerted by
  growth factors that specifically overcome
  inhibitions at cell cycle checkpoints, especially
  G1.
• Binding of growth factors to their cell surface
  receptors initiates a cascade of reactions that
  induces two class of genes.

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Signal Transduction

• Early response genes (e. g. protooncogenes jun,
  fos, myc) are rapidly activated (15 min).
• Protooncogene genes are genes that if mutated,
  can promote carcinogenesis.
• Each of the jun and fos families codes for a
  series of transcription factors containing
  leucine zipper domains.
• The function of the myc family is not understood
  but is critically important in normal cell
  function.

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Signal Transduction

• Delayed response genes are induced by activities
  of the early response phase.
• Among the products are the Cdks, the cyclins, and
  other factors needed for cell division.
• Epidermal growth factor (EGF) is an example.
  Its role in the activation of the transcription
  factor AP-1 is shown on the next slide.

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Fig 18.33