Ch' 19 Transcription and RNA

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Ch. 19 Transcription and RNA

• Transcription transfer of genetic information
  from DNA to RNA
• Gene a segment of DNA that is transcribed for
  the purpose of expressing the encoded genetic
  information or transforming it to a form that is
  more useful to the cell
• 1. protein-coding genes
• 2. One polypeptide vs multi polypeptide (operon)
• 3. RNA processing (intron)
• 4. Regulatory DNA sequences

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Figure 19.01 Genes in a segment of human
chromosome 20.
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• Human genome 30,000-35,000genes, average length
  of about 27,000 bp
• Exon average length of about 145 bp
• Intron average length of about 3365 bp
• ORF (Open Reading Frame) DNA sequence can be
  translated, but its protein products not
  identified
• ESTs (Expressed Sequence Tags) may represent (a
  part of) novel genes
• Number of genes vs organismal complexity?

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• Transcription Initiation
• RNA transcription carried out by immobile
  protein complex that reel in the DNA (distinct
  from DNA replication sites)
• ? locating the transcription site
• ? melting apart the double-
• stranded DNA
• ? initiating RNA synthesis
• ? extending the RNA molecule
• ? termination of RNA synthesis

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• Transcription begins at promoters
• promoter a site recognized by specific proteins
  (RNA polymerase or its interacting proteins) to
  begin RNA synthesis
• 1) bacteria a sequence of about 40 bases on the
  5 sides of the transcription start site
• ? two consensus sequences -35 -10
• ? s subunit of RNA polymerase recognizes and
  binds to the promoter site (recruites RNA
  polymerase)
• 2) eukaryotes most promoters contain TATA box
• ? TATA box AT rich seq. just upstream of the
  5 sides of the
• transcription initiation sites
  
• ? other additional seq. gthundreds bp upstream
• ? recruite RNA polymerase by a series of complex
  protein-protein interactions

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Figure 19.03 The E. coli promoter.
Figure 19.04 A eukaryotic promoter.
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• Transcription factors recognize eukaryotic
  promoters
• Sequence specificity
• Accessibility chromatin condensation state
  (silent/active) and regulated by histone
  modification (histone code)
• ? histone acetylation histone
  acetyltransferase/ histone deacetylase
• ? general transcription factors required for
  transcription initiation, TFIIA, B, D, E, F and H
• TFIID TBP (TATA binding proteins) and 12
  additional TAFs (TBP associated factors, subunits
  for promoter specificity)
• ? TBP binds to the TATA box of a eukaryotic
  promoter
• introduces two sharp kinks into
  DNA

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Transcription factors recognize eukaryotic
promoters 2. Accessibility ? TAFs15- 250 kD,
promoter specificity was formed by combination of
TAFs (about 7 or 8 TAFs for a TFIID complex),
receiving regulatory signals TAFII250
kinase, histone acetyltransferase and
ubiquitin ligase functions Bromo
domain a hydrophobic pocket to bind to
acethylated Lys. 3. Recruiting of other general
transcription factors TBP- DNA complex ? TFIIA
TFIIB ? other factors (TFIIH helicase) ?
formation of a transcription bubble a docking
site for RNA polymerase, about 50 proteins
(including 12 subunits of RNA pol)
TATA-less promoter
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Figure 19.05 Structure of TBP bound to DNA.
Model of eukaryotic transcription factors bound
to DNA.
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• Enhancers and silencers act as a distance from
  the promoter
• ratio of the most and least expressed genes
  prokaryotes 1000, eukaryotes109 ? how to
  control? Enhancers!!
• Enhancers (Silencers) DNA sequences for binding
  of proteins (activator or repressor) to regulate
  transcription of the target genes
• - requires DNA looping for interaction of
  enhancer-binding proteins
• with promoter-binding proteins
• 3. Changes in gene expression accomplished
  through transcription-regulatory proteins
  (regulated by signal-transduction pathway or
  other mechanisms)
• example Glucocoritoid receptor
• STAT

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Figure 19.09 Overview of enhancer function.
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Glucocoritoid receptor
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STAT (signal transducer and activator of
transcription)
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Box 19.A DNA binding protein (motif)
helix-turn-helix zinc-finger
(motif) leucine zipper
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• Some genes are coordinately expressed
• Prokaryotes
• operon (the organization of functionally
  related genes) ensure coordiante expression in
  response to some metabolic signal
• example trp operons
• 2. eukaryotes not a physical cluster
• synexpression groups a group of coregulated
  genes by sharing similar control seq. and
  regulatory proteins (5-10 of genes)
• ?? promote efficient transcription
• ? ensure the stoichiometric synthesis of
  related gene products

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Box 19. B. lac operon
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Box 19. B. lac operon
Repressor tetramer bind to 2 of 3 lac operator
seq. and prevent transcription initiation
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• 2. RNA polymerase
• Number 1 for bacteria,
• 32 for eukaryotes
• eukaryotic RNA pol I
• RNA pol II
• RNA pol III

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• 2. RNA polymerase
• Number
• RNA pol II
• ? gt500kD, a hand structure like DNA pol
• ? conserved seq. among eukaryotes identical
  structures
• ? Requires two Mg ions
• ? Catalyzes nucleophilic attack of the 3 OH of
  the growing polynucleotide chain ?extends in the
  5?3 direction
• ? No primer is needed
• ? DNA-RNA hybrid double helix (8-9 base pairs)
• ? transcription bubble 12-14 nucleotides

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Figure 19.14a RNA polymerase with bound DNA and
RNA.
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• RNA polymerase is a processive enzyme
• clamp rotates over the DNA template and the RNA
  transcript, ensure the processivity of the enzyme
• helix oscillates between a straight and bent
  conformation help to add new ribonucleotide to
• the growing transcript

• Proofreading ability
• i) mismatch cause distorted double helix
  and polymerization is stopped
• ii) the transcript backs out and nuclease
  (TFIIS) cleaves the mismatched base
• iii) may resume polymerization
• ? The transcription bubble size and DNA-RNA
  double helix length constant during
  polymerization

Figure19.15 Schematic view of backtracking RNA
in RNA polymerase.
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• What allows RNA polymerase to elongate a
  transcript?
• Repeat of the initiation before elongation a
  puzzle
• RNA pol. Initiation mode ? elongation
  mode
• The switch C-terminal domain of RNA pol II
  contains 52 repeats (Y-S-P-T-S-P-S)
• i) initiation non-phosphorylated
• ii) elongation phosphorylated by TFIIH or
  cdk
• After elongation mode of one pol II, another pol
  II is recruited to the initiation site by the
  remaining general transcription factor complex in
  the promoter

Transition from transcription initiation to
elongation
Transcription through a nucleosome DNA
never entirely leaves the histone octamer .
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• What allows RNA polymerase to elongate a
  transcript?
• 1) Repeat of the initiation before elongation
• 2) elongation elongator (6 protein complex)
  binds to phosphorylated C-terminal of RNA pol II,
  accelerates transcription
• TFIIH and TFIIF remained
• P-CTD of polII docking site for RNA processing
  proteins
• ? during elongation, pol II pause or cease
  transcription (in response to certain DNA
  signals)
• 3) termination
• ? E. coli about half of transcription sites a
  base-paired hairpin followed by a series of U
  residues ?destabilizes RNA pol
• ? eukaryotes no such termination signal, how?

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3. RNA processing affect mRNAs ability to
direct translation 1) Life cycle of mRNA
transcription ? covalent modification of both
ends ? splicing to remove introns ? export from
the nucleus ? delivery to ribosomes ? degradation
of the mRNAs 2) Processing begins just after
emerging of the transcript from RNA pol 3)
Processing promotes elongation 4) Processing
enzymes recruited through phosphorylated
C-terminal of pol II 5) Processing is closely
linked (coupled) to transcription
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3. RNA processing 5 capping Cap
7-methylguanosine, three different enzymes
Three enzymes Triphosphatase
Guanylyltransferase Methyltransferase 3
polyadenylation Splicing mRNA turnover
Figure 19.18 An mRNA 5 cap.
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5 Capping of mRNA
RNA triphosphatase
In mammals, single bifunctional capping enzyme
RNA guanylyl transferase
RNA methyltransferase
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• 3. RNA processing
• 5 capping
• 3 polyadenylation
• Poly A signal (AAUAAA) a signal for a protein
  poly (A) polymerase to cleave the transcript
  and extend it by adding adenosine residues (poly
  A tail), no template need
• Multiple poly A tail binding proteins protect
  the 3 end of mRNA from 3 exonuclease attack
• may serve as a handle for the proteins that
  deliver mRNA to ribosomes
• 20-50 poly As? (average 250 poly As)

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• 3. RNA processing
• 5 capping
• 3 polyadenylation
• splicing
• Removal of an intervening sequence (intron) and
  joining of the two adjacent expressed sequences
  (exons)
• Spliceosome a complex of five small RNA(snRNAs)
  molecules and their associated proteins (gt50)
• Recognize conserved seq. at the 5 intron/exon
  junction and A at the branch points

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3. RNA processing splicing 4) A two step
transesterification process
Figure 19.21 mRNA splicing.
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• 3. RNA processing
• 5 capping
• 3 polyadenylation
• Splicing
• 5) Some introns in protozoan rRNA genes
  self-splicing without aid of proteins (Ribozymes)
• 6) yeast one small intron for a few genes
• most mammalian genes 90 of the genes are
  non-coding seq.
• 7) Origin of intron
• ? Remnants of mobile genetic elements
  (transposons) splicesome origin?
• RNA molecules self-splicing, reverse
  transcription and insertion into genome
• 8) A mechanism to promote variation of gene
  expression 60 of human genes exhibit splicing
  one gene/ gt1 polypeptide

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Figure 19.22 Alternative splicing.
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• 3. RNA processing
• mRNA turnover
• mRNA 5 of total cellular RNA, continuous
  synthesis and degradation
• lifetime of mRNA another regulation aspect of
  gene expression, ? lt1hr 24 hr
• depend on degradation speed of poly A tail
  (depends on signal in 3 UnTranslated Region)
• 3) Probably always binds to RNA-binding proteins
  during transport from nucleus to ribosomes or
  other regions
• 4) Addressing system (seq. or 2 st. of mRNA)
  determine ultimate cytosolic destination
• 5) Poly A tail shortening ?decapping ?
  exonuclease acceess to 5 end ? destruction of
  the entire message
• 6) RNA-binding regulatory proteins monitoring
  RNA integrity (mRNA with premature stop codon,
  RNA interference)

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Box 19-C. RNA interference
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Figure 19.23 mRNA decay.
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• rRNA and tRNA processing
• rRNA (ribosomal RNA)
• ?synthesized by Pol I
• ?Maturation in nucleolus
• ?initial eukaryotic rRNA transcript ?processed
  to 3 rRNA moleulces (28S, 18S, 5.8S)
• ? Base modification of bases
• pseudouridine
• Methylation guided by small nucleolar RNA
  snoRNAs to recognize and pair with specific 15-
  bases in rRNA, and then directing the associated
  methylase to each site)

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• rRNA and tRNA processing
• rRNA
• Synthesis speed 7500 rRNA transcripts/minute in
  a rapidly growing cells, each associated with
  about 150 different snoRNAs
• ? functional ribosomes 3 rRNAs with 80 different
  proteins, needs careful coordination
• 2) tRNA

? synthesized by Pol III ? pre tRNA i)
eukaryotes nucleotide processing, covalent
modification of bases ii) prokaryotes
trimmed by RNase P ribozyme ? covalent
modification 10-25 bases ? splicing ?
Addition of CCA at the 3 end maturation (amino
acid attachment site)
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Figure 19.25 Some modified nucleotides in tRNA
molecules.
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• 4. RNA A versatile macromolecule
• Catalytic RNA complex tertiary structure and
  reactive functional groups (like proteins)
• single stranded RNA can adopt highly convoluted
  shapes through base-pairing
• nonstandard base pairs and hydrogen bonding
  interactions among three bases
• base stacking stability
• First ribozyme the hammerhead ribozyme
• RNA world
• protein cofactors to enhance its catalytic
  activity

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Figure 19.26 Some nonstandard base pairs in RNA.
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Figure 19.27a The hammerhead ribozyme.