Transcription

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• Transcription

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Central Dogma
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Genes

• Sequence of DNA that is transcribed.
• Encode proteins, tRNAs, rRNAs, etc..
• Housekeeping genes encode proteins or RNAs that
  are essential for normal cellular activity.
• Simplest bacterial genomes contain 500 to 600
  genes.
• Mulitcellular Eukaryotes contain between 15,000
  and 50,000 genes.

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Types of RNAs

• tRNA, rRNA, and mRNA
• rRNA and tRNA very abundant relative to mRNA.
• But mRNA is transcribed at higher rates than rRNA
  and tRNA
• Abundance is a reflection of the relative
  stability of the different forms of RNA

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RNA Content of E. coli Cells
type Steady State Levels Synthetic Capacity Stability
rRNA 83 58 High
tRNA 14 10 High
mRNA 3 32 Very Low
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Phases of Transcription

• Initiation Binding of RNA polymerase to
  promoter, unwinding of DNA, formation of primer.
• Elongation RNA polymerase catalyzes the
  processive elongation of RNA chain, while
  unwinding and rewinding DNA strand
• Termination termination of transcription and
  disassemble of transcription complex.

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E. Coli RNA Polymerase

• RNA polymerase core enzyme is a multimeric
  protein a2,b, b, w
• The b subunit is involved in DNA binding
• The b subunit contains the polymerase active site
• The a subunit acts as scaffold on which the other
  subunits assemble.
• Also requires s-factor for initiation forms holo
  enzyme complex

Site of DNA binding and RNA polymerization
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s-factor

• The s-factor is required for binding of the RNA
  polymerase to the promoter
• Association of the RNA polynerase core complex w/
  the s-factor forms the holo-RNA polymerase
  complex
• W/o the s-factor the core complex binds to DNA
  non-specifically.
• W/ the s-factor, the holo-enzyme binds
  specifically with high affinity to the promoter
  region
• Also decreases the affinity of the RNA polymerase
  to non-promoter regions
• Different s-factors for specific classes of genes

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General Gene Structure

• Promoter sequences recognized by RNA polymerase
  as start site for transcription.
• Transcribed region template from which mRNA is
  synthesized
• Terminator sequences signaling the release of
  the RNA polymerase from the gene.

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Gene Promoters

• Site where RNA polymerase binds and initiates
  transcription.
• Gene that are regulated similarly contain common
  DNA sequences (concensus sequences) within their
  promoters

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Important Concensus Sequences

• Pribnow Box position 10 from transcriptional
  start
• -35 region position 35 from transcriptional
  start.
• Site where s70-factor binds.

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Other s-Factors

• Standard genes s70
• Nitrogen regulated genes s54
• Heat shock regulated genes s32

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How does RNA polymerase finds the promoter?

• RNA polymerase does not disassociate from DNA
  strand and reassemble at the promoter (2nd order
  reaction to slow)
• RNA polymerase holo-enzyme binds to DNA and scans
  for promoter sequences (scanning occurs in only
  one dimension, 100 times faster than diffusion
  limit)
• During scanning enzyme is bound non-specifically
  to DNA.
• Can quickly scan 2000 base pairs

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Transcriptional Initiation

• Rate limiting step of trxn.
• Requires unwinding of DNA and synthesis of
  primer.
• Conformational change occurs after DNA binding of
  RNA polymerase holo-enzyme.
• First RNA Polymerase binds to DNA
  (closed-complex), then conformational change in
  the polymerase (open complex) causes formation of
  transcription bubble (strand separation).

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Initiation of Polymerization

• RNA polymerase has two binding sites for NTPs
• Initiation site prefers to binds ATP and GTP
  (most RNAs begin with a purine at 5'-end)
• Elongation site binds the second incoming NTP
• 3'-OH of first attacks alpha-P of second to form
  a new phosphoester bond (eliminating PPi)
• When 6-10 unit oligonucleotide has been made,
  sigma subunit dissociates, completing
  "initiation
• NusA protein binds to core complex after
  disassociation of s-factor to convert RNA
  polymerase to elongation form.

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Transcriptional Initiation
Closed complex
Open complex
Primer formation
Disassociation of s-factor
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Chain Elongation

• Core polymerase - no sigma
• Polymerase is accurate - only about 1 error in
  10,000 bases
• Even this error rate is OK, since many
  transcripts are made from each gene
• Elongation rate is 20-50 bases per second -
  slower in G/C-rich regions (why??) and faster
  elsewhere
• Topoisomerases precede and follow polymerase to
  relieve supercoiling

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Transcriptional Termination

• Process by which RNA polymerase complex
  disassembles from 3 end of gene.
• Two Mechanisms Pausing and rho-mediated
  termination

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Pausing induces termination

• RNA polymerase can stall at pause sites
• Pause sites are GC rich (difficult to unwind)
• Can decrease trxn rates by a factor of 10 to 100.
• Hairpin formation in RNA can exaggerate pausing
• Hairpin structures in transcribed RNA can
  destabilize DNARNA hybrid in active site
• Nus A protein increases pausing when hairpins
  form.

3end tends to be AU rich easily to disrupt
during pausing. Leads to disassembly of RNA
polymerase complex
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Rho Dependent Termination

• rho is an ATP-dependent helicase
• it moves along RNA transcript, finds the
  "bubble", unwinds it and releases RNA chain

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Eukaryotic Transcription

• Similar to what occurs in prokaryotes, but
  requires more accessory proteins in RNA
  polymerase complex.
• Multiple RNA polymerases

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Eukaryotic RNA Polymerases
type Location Products
RNA polymerase I Nucleolus rRNA
RNA polymerase II Nucleoplasm mRNA
RNA polymerase III Nucleoplasm rRNA, tRNA, others
Mitochondrial RNA polymerase Mitochondria Mitochondrial gene transcripts
Chloroplast RNA polymerase Chloroplast Chloroplast gene transcripts
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Eukaryotic RNA Polymerases

• RNA polymerase I, II, and III
• All 3 are big, multimeric proteins (500-700 kD)
• All have 2 large subunits with sequences similar
  to ? and ?' in E.coli RNA polymerase, so
  catalytic site may be conserved

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Eukaryotic Gene Promoters

• Contain AT rich concensus sequence located 19 to
  27 bp from transcription start (TATA box)
• Site where RNA polymerase II binds

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RNA Polymerase II

• Most interesting because it regulates synthesis
  of mRNA
• Yeast Pol II consists of 10 different peptides
  (RPB1 - RPB10)
• RPB1 and RPB2 are homologous to E. coli RNA
  polymerase ? and ?'
• RPB1 has DNA-binding site RPB2 binds NTP
• RPB1 has C-terminal domain (CTD) or PTSPSYS
• 5 of these 7 have -OH, so this is a hydrophilic
  and phosphorylatable site

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More RNA Polymerase II

• CTD is essential and this domain may project away
  from the globular portion of the enzyme (up to 50
  nm!)
• Only RNA Pol II whose CTD is NOT phosphorylated
  can initiate transcription
• TATA box (TATAAA) is a consensus promoter
• 7 general transcription factors are required

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Transcription Factors

• Polymerase I, II, and III do not bind
  specifically to promoters
• They must interact with their promoters via
  so-called transcription factors
• Transcription factors recognize and initiate
  transcription at specific promoter sequences

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Transcription Factors

• TFAIIA, TFAIIB components of RNA polymerase II
  holo-enzyme complex
• TFIID Initiation factor, contains TATA binding
  protein (TBP) subunit. TATA box recognition.
• TFIIF (RAP30/74) decrease affinity to
  non-promoter DNA

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Eukaryotic Transcription

• Once initiation complex assembles process similar
  to bacteria (closed complex to open complex
  transition, primer formation)
• Once elongation phase begins most transcription
  factor disassociate from DNA and RNA polymerase
  II (but TFIIF may remain bound).
• TFIIS Elongation factor binds at elongation
  phase. May also play analogous role to NusA
  protein in termination.

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• Transcriptional Regulation and
• RNA Processing

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Gene Expression

• Constitutive Genes expressed in all cells
  (Housekeeping genes)
• Induced Genes whose expression is regulated by
  environmental, developmental, or metabolic
  signals.

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Regulation of Gene Expression
RNA Processing
mRNA
RNA Degradation
AAAAAA
5CAP
Active enzyme
Post-translational modification
Protein Degradation
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Transcriptional Regulation

• Regulation occurring at the initiation of
  transcription.
• Involves regulatory sequences present within the
  promoter region of a gene (cis-elements)
• Involves soluble protein factors (trans-acting
  factors) that promote (activators) or inhibit
  (repressors) binding of the RNA polymerase to the
  promoter

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Cis-elements

• Typically found in 5 untranscribed region of the
  gene (promoter region).
• Can be specific sites for binding of activators
  or repressors.
• Position and orientation of cis element relative
  to transcriptional start site is usually fixed.

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Enhancers

• Enhancers are a class of cis-elements that can be
  located either upstream or downstream of the
  promoter region (often a long distance away).
• Enhancers can also be present within the
  transcribed region of the gene.
• Enhancers can be inverted and still function
• 5-ATGCATGC-3 5-CGTACGTA-3

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Two Classes of Trans-Acting Factors

• Activators and repressors- Bind to cis-elements.
• Co-activators and co-repressors bind to
  proteins associated with cis-elements. Promote or
  inhibit assembly of transcriptional initiation
  complex

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Structural Motifs in DNA-Binding Regulatory
Proteins

• Crucial feature must be atomic contacts between
  protein residues and bases and sugar-phosphate
  backbone of DNA
• Most contacts are in the major groove of DNA
• 80 of regulatory proteins can be assigned to one
  of three classes helix-turn-helix (HTH), zinc
  finger (Zn-finger) and leucine zipper (bZIP)
• In addition to DNA-binding domains, these
  proteins usually possess other domains that
  interact with other proteins

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The Helix-Turn-Helix Motif

• contain two alpha helices separated by a loop
  with a beta turn
• The C-terminal helix fits in major groove of DNA
  N-terminal helix stabilizes by hydrophobic
  interactions with C-terminal helix

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The Zn-Finger Motif
Zn fingers form a folded beta strand and an alpha
helix that fits into the DNA major groove.
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The Leucine Zipper Motif

• Forms amphipathic alpha helix and a coiled-coil
  dimer
• Leucine zipper proteins dimerize, either as homo-
  or hetero-dimers
• The basic region is the DNA-recognition site
• Basic region is often modeled as a pair of
  helices that can wrap around the major groove

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Binding of some trans-factors is regulated by
allosteric modification
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Transcription Regulation in Prokaryotes

• Genes for enzymes for pathways are grouped in
  clusters on the chromosome - called operons
• This allows coordinated expression
• A regulatory sequence adjacent to such a unit
  determines whether it is transcribed - this is
  the operator
• Regulatory proteins work with operators to
  control transcription of the genes

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Induction and Repression

• Increased synthesis of genes in response to a
  metabolite is induction
• Decreased synthesis in response to a metabolite
  is repression

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lac operon

• Lac operon encodes 3 proteins involved in
  galactosides uptake and catabolism.
• Permease imports galactosides (lactose)
• b-galactosidase Cleaves lactose to glucose and
  galactose.
• b-galactoside transacetylase acetylates
  b-galactosides
• Expression of lac operon is negatively regulated
  by the lacI protein

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The lac I protein

• The structural genes of the lac operon are
  controlled by negative regulation
• lacI gene product is the lac repressor
• When the lacI protein binds to the lac operator
  it prevents transcription
• lac repressor 2 domains - DNA binding on
  N-term C-term. binds inducer, forms tetramer.

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Inhibition of repression of lac operon by inducer
binding to lacI

• Binding of inducer to lacI cause allosteric
  change that prevents binding to the operator
• Inducer is allolactose which is formed when
  excess lactose is present.

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Catabolite Repression of lac Operon (Positive
regulation)

• When excess glucose is present, the lac operon is
  repressed even in the presence of lactose.
• In the absence of glucose, the lac operon is
  induced.
• Absence of glucose results in the increase
  synthesis of cAMP
• cAMP binds to cAMP regulatory protein (CRP) (AKA
  CAP).
• When activated by cAMP, CRP binds to lac promoter
  and stimulates transcription.

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Post-transcriptional Modification of RNA

• tRNA Processing
• rRNA Processing
• Eukaryotic mRNA Processing

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tRNA Processing

• tRNA is first transcribed by RNA
• Polymerase III, is then processed
• tRNAs are further processed in the chemical
  modification of bases

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rRNA Processing

• Multiple rRNAs are originally transcribed as
  single transcript.
• In eukaryotes involves RNA polymerase I
• 5 endonuclases involved in the processing

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Processing of Eukaryotic mRNA
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5 Capping

• Primary transcripts (aka pre-mRNAs or
  heterogeneous nuclear RNA) are usually first
  "capped" by a guanylyl group
• The reaction is catalyzed by guanylyl transferase
  
• Capping G residue is methylated at 7-position
• Additional methylations occur at 2'-O positions
  of next two residues and at 6-amino of the first
  adenine
• Modification required to increase mRNA stability

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3'-Polyadenylylation

• Termination of transcription occurs only after
  RNA polymerase has transcribed past a consensus
  AAUAAA sequence - the poly(A) addition site
• 10-30 nucleotides past this site, a string of 100
  to 200 adenine residues are added to the mRNA
  transcript - the poly(A) tail
• poly(A) polymerase adds these A residues
• poly(A) tail may govern stability of the mRNA

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Splicing of Pre-mRNA

• Pre-mRNA must be capped and polyadenylated before
  splicing
• In "splicing", the introns are excised and the
  exons are sewn together to form mature mRNA
• Splicing occurs only in the nucleus
• The 5'-end of an intron in higher eukaryotes is
  always GU and the 3'-end is always AG
• All introns have a "branch site" 18 to 40
  nucleotides upstream from 3'-splice site

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Splicing of Pre-mRNA

• Lariat structure forms by interaction with
  5splice site G and 2OH of A in the branch site.
• Exons are then joined and lariot is excised.
• Splicing complex includes snRNAs that are
  involved in identification of splice junctions.