GEN 272 Introductory Molecular Genetics

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GEN 272Introductory Molecular Genetics

• Lecture Six

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REMINDER!

• First test Wednesday, 27 February 2008 (during
  lecture period)

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Previously

• The genetic code governs the synthesis of
  proteins from mRNA, where each building block of
  proteins (i.e. amino acid) is made from a codon
  (three ribonucleotides in mRNA) the triplet
  nature of the code
• Any ribonucleotide along the mRNA molecule
  belongs to a single codon nonoverlapping nature
  of the code. However, a single mRNA sequence may
  have several initiation points for protein
  synthesis overlapping genes
• The genetic code is degenerate (Wobble
  hypothesis) the first 2 positions of the codon
  are critical whereas the third position is less
  constrained
• The code is nearly universal (and ordered) for
  example, amino acids may be encoded by different
  codons in different genomes

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Transcription on a DNA template

• Transcription is a process by which RNA molecules
  are synthesized on a DNA template
• it results in the synthesis of an mRNA molecule
  that is complementary to one of the two DNA
  strands
• each triplet codon in the mRNA is complementary
  to the anticodon region of its corresponding tRNA
  region
• the significance of transcription is enormous
  because it is the initial step in the process of
  information flow within the cell
• That RNA is an intermediate molecule in the
  central dogma is suggested by observations that
• DNA does not appear to participate directly in
  protein synthesis (DNA and protein occur in
  different locations in the cell)
• RNA (chemically similar to DNA) is synthesized in
  the nucleus of eukaryotic cells, where DNA is
  also found
• following synthesis, most RNA migrates to the
  cytoplasm where protein synthesis occurs
• the amount of RNA is generally proportional to
  the amount of protein in a cell

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RNA polymerase

• RNA polymerase is the enzyme known to direct RNA
  synthesis
• similar to DNA polymerase, it has the same
  general requirements except that the substrate
  nucleotides contain ribose instead of deoxyribose
  form of the sugar
• however, unlike DNA polymerase, no primer is
  required to initiate synthesis
• RNA polymerase from E. coli has been extensively
  characterized and consists of subunits designated
  ?, ?, ?, ?, and ?
• holoenzyme (active form) contains subunits ?2???
  (500 000 Da)
• ? and ? polypeptides provide the catalytic
  
  basis and active site for transcription
• the ? subunit plays a regulatory function
  
  involving the initiation of RNA
  
  transcription
• note although there is a single form of
  
  the enzyme in E. coli, there are several
  different ? factors that create variations of
  the polymerase holoenzyme
• eukaryotes display three distinct forms of RNA
  pol, each consisting of a greater number of
  polypeptide subunits

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Promoters, Template Binding and the ? subunit

• Note we shall refer to the template strand as
  the DNA strand that is being transcribed and the
  nontemplate strand as the strand that is
  complementary to the template strand
• The initial step of transcription involves
• ? subunit of RNA polymerase recognize specific
  DNA sequences called promoters (process termed
  template binding). Promoters are regions located
  on the 5 region (also called upstream) from the
  point of initial transcription of a gene
• RNA pol scans the promoter region until it
  recognize the specific target sequence and binds
  to 60 bp (base pairs), of which 40 bp are
  upstream from the point of initial transcription
• following binding, the helix is denatured or
  unwound locally making the template strand
  accessible to the enzyme's transcription
  machinery
• note the point at which transcription actually
  begins is called the transcription start site
• Promoter sequences are important as they govern
  the initiation of transcription
• mutations in promoter sequences may have the
  effect of reducing or enhancing the initiation of
  gene expression (for example, by affecting RNA
  pol binding)

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Terminology

• Consensus sequences
• sequences that share homology in different genes
  of the same organism or in one or more genes of
  related organisms
• note their conservation throughout evolution
  attests to their critical nature in biological
  processes
• Two consensus sequences found in bacterial
  promoter regions are
• TATAAT sequence located 10 nucleotides upstream
  from the site of initial transcription (also
  called the -10 region or the Pribnow box)
• TTGACA sequence located 35 nucleotides upstream
  from the site of initial transcription (also
  called the -35 region)
• The -10 and -35 regions are referred to as
  cis-acting elements (cis next to)
• In contrast, trans-acting elements are molecules
  that bind to cis-elements (trans across from)

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Initiation and Elongation

• Following template binding, RNA polymerase
  catalyzes transcription initiation, whereby the
  first 5-ribonucleoside triphosphate is inserted
• the inserted 5-ribonucleoside triphosphate is
  complementary to the first nucleotide at the
  start of site of the DNA template strand
• note unlike replication, no primer is required
  for initiation
• RNA polymerization proceeds with more
  ribonucleotide complements being added (via the
  formation of phosphodiester bonds) in the 5 to
  3 direction, resulting in the formation of a
  temporary 8 bp DNA/RNA duplex whose chains run in
  opposite directions
• After the insertion of these nucleotides, the ?
  subunit will dissociate from the holoenzyme and
  chain elongation proceeds under the direction of
  the core enzyme

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Initiation and Elongation
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Termination

• Eventually, the enzyme transverses the entire
  gene until it encounters specific nucleotide
  sequences (40 bp) that act as a termination
  signal
• in bacteria, genes are in close proximity that
  the end of one gene is adjacent to the upstream
  region of the next gene
• the termination sequence is also transcribed into
  RNA where it folds back on itself to form a
  hairpin secondary structure
• also involved in the termination process is the
  termination factor, rho (?) protein
• When termination is achieved, RNA polymerase will
  cease to add ribonucleotides and dissociates from
  the DNA template
• at the end of transcription, there will be an RNA
  molecule that is precisely complementary to the
  template strand of a gene (note T is replaced by
  U in RNA)
• In bacteria, groups of genes whose protein
  products are involved in the same metabolic
  pathway tend to be clustered together along a
  chromosome
• hence, (considering the close proximity of genes)
  a large mRNA is produced (called a polycistronic
  mRNA)
• in eukaryotes, mRNAs are monocistronic

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Eukaryotic Transcription

• Most of the general aspects of transcription in
  eukaryotes are similar to that in prokaryotes.
  However, notable exceptions (in eukaryotes)
  include,
• transcription occurs within the nucleus, under
  the direction of three separate RNA pol enzymes
  (note translation occurs in the cytoplasm)
• chromatin remodeling complex conformational
  change that involves the uncoiling of the compact
  chromatin fiber that includes nucleosomes
• transcription and regulation of transcription
  involves a more extensive interaction between
  cis-acting sequences and trans-acting protein
  factors. In addition, enhancers (located in the
  5 regulatory region) may also be involved
• eukaryotic mRNA has to be processed before
  translation. Sometimes called pre-mRNA, they are
  part of a group of molecules collectively called
  heterogeneous nuclear mRNA (hnRNA), which complex
  with proteins to form heterogeneous nuclear
  ribonucleoprotein particles (hnRNPs). hnRNAs are
  further edited (gene splicing) to produce the
  final product that shall be translated

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Transcription Initiation in Eukaryotes

• Eukaryotic RNA pol exists in three unique forms,
  each of which transcribes different types of
  genes
• RNA pol II (RNP II) is regulated
  by
  both cis-acting elements in the
  gene
  itself, and by a number of
  factors that
  bind to these elements
• the first is the core-promoter
  
  element determines where RNP II binds to the
  DNA and where it begins copying DNA into RNA
• the other two regulatory DNA sequences are the
  promoter and enhancer elements influence the
  efficiency or the rate of transcription by RNA
  pol II as the process proceeds
• note in eukaryotes, promoter refer to a
  collection of cis-acting DNA elements including
  the core-promoter sequence (where RNA pol II
  binds) and the promoter and enhancer elements
  that influence RNP II activity

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Regulatory DNA Elements

• Goldberg-Hogness or TATA box (cis-acting)
  present in almost all eukaryotic genes
• located about 30 nucleotide pairs upstream (-30)
  from the start point of transcription
• TATA boxes share a consensus heptanucleotide
  sequence consisting solely of A and T residues
  (TATAAAA). note the sequence is functionally
  analogous to the -10 region of prokaryotic genes
• TATA box is the site where RNP II binds to the
  DNA template and transcription typically begins
  25 to 30 bp downstream
• thus, the TATA box determines where transcription
  begins (i.e. transcription start site)
• CAAT box (cis-acting)
• contains the consensus sequence GGCCAATCT, which
  is typically located within 100 nucleotides
  upstream of the start of transcription
• together with other types of promoter elements
  also located upstream of the transcription start
  site, CAAT box influence the efficiency of the
  promoter

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More regulatory DNA elements

• Enhancers (cis-acting)
• locations vary, though they are often located
  even farther upstream than the regions already
  mentioned or even downstream or within the gene
  itself
• though not directly involved in RNP II binding to
  the core promoter, they are essential to highly
  efficient initiation of transcription
• Transcription Factors, TFs (trans-acting)
  facilitate RNP II binding and, therefore,
  transcription initiation. There are two broad
  categories,
• general TFs absolutely required for all RNP II
  mediated transcription
• specific TFs influence the efficiency or the
  rate of RNP II transcription
• General TFs are essential because RNP II cannot
  bind directly to eukaryotic promoter sites and
  initiate transcription without their presence
• for example, in humans, general TFs have been
  characterized and designated TFIIA, TFIIB, etc
• TFIID binds directly to the TATA-box via the
  TATA-binding protein (TBP). Upon its binding,
  several other general TFs are recruited to form
  an extensive pre-initiation complex, which is
  then bound by RNP II

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RNP II during transcription

• The process has been detailed by Roger Kornberg
  and colleagues from their studies in yeast
• in yeast, RNP II contains two large subunits and
  10 smaller ones forming a huge 3D complex of
  about 500 kDa
• the promoter region of the DNA duplex to be
  transcribed enters a positively charged cleft
  between the two large subunits that resemble a
  pair of jaws
• prior to association with DNA, the cleft is open.
  Upon association with DNA, the cleft closes
  partially to secure the duplex during the
  initiation of transcription
• once secured by the clamp (region of the enzyme
  involved in transcription), a small duplex region
  of DNA separates at the active center, where
  complementary RNA synthesis is initiated
• however, the complex remains unstable in that
  transcription terminates only after the
  incorporation of a few ribonucleotides (abortive
  transcription)
• the process is repeated a few times until a
  stable RNADNA hybrid is formed (11 nucleotides
  long)
• at this point, abortive transcription is overcome
  and a stable complex is achieved and
  transcription proceeds in earnest (i.e. a level
  of highly processive RNA polymerization)
• the growing RNA chain runs through a groove in
  the enzyme and exits at the lid, situated at the
  top and back of the enzyme. At the bottom and
  back, there is a pore through which RNA
  precursors enter
• eventually termination signals are encountered,
  the complex becomes unstable, the clamp open, and
  both DNA and RNA are released as transcription is
  terminated

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hnRNA Processing

• Note in prokaryotes, there is a direct
  relationship between DNA and RNA, i.e. the DNA
  base sequence is transcribed into mRNA which is
  then immediately translated into protein
• In eukaryotes, the complex processing of mRNA
  occurs before it is transported to the cytoplasm
  to participate in translation
• The initial proposition came from observations by
  James Darnell and colleagues that large
  heterogeneous nuclear RNA (hnRNA) in mammalian
  nuclei contained nucleotide sequence common to
  smaller mRNA molecules present in the cytoplasm
  (see Fig. 13-10)
• they proposed that the initial transcript of a
  gene results in a large RNA molecule that must
  first be processed in the nucleus before it
  appears in the cytoplasm as a mature mRNA
  molecule
• Posttranscriptional modification of eukaryotic
  RNA transcripts involves
• the addition of a 7-methylguanosine (7-mG) cap on
  the 5-end of the RNA molecule
• the addition of poly-A sequences on the 3-end of
  the molecule, and
• the removal of intervening, non-coding sequences

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Take Home Message

• Transcription is directed by RNA polymerase
• the ? subunit is the main role playing subunit in
  the enzyme isoform of prokaryotes
• of the three isoforms in eukaryotes, RNP II
  produces mRNA
• RNA pol acts through the recognition of specific
  DNA sequences (promoters) located upstream of the
  transcription initiation site. In addition,
  cis-acting elements (and trans-acting elements in
  eukaryotes) exists in DNA sequences that enhance
  RNA polymerase binding and transcription efficacy
• In eukaryotes, posttranscriptional modifications
  are necessary to prepare mRNA molecules for
  translation in the cytoplasm

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