Mechanisms of transcription

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Mechanisms of transcription

• By ZhaoYi
• ??????
• 200431060015

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• After having discussed the maintenance of genome,
  we now turn to the question of how that genetic
  material is expressed.

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• Transcription is somewhat similar to DNA
  replication. Both involve enzymes that synthesize
  a new strand of nucleic acid complementary to a
  DNA template strand.

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The differences are

• 1gt.RNA polymerase
• it does not need a primer rather, it can
  initiate transcription de novo.

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RNA polymerase
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• DNA-dependent RNA polymerases are responsible for
  building RNA transcripts (mRNA, tRNA, rRNA)
  complementary to template strands of double
  stranded DNA, and regulation of their activity is
  often the final step in cellular pathways that
  control the expression of genes.

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RNA Polymerase Structure

• The massive holoenzyme contains 6 subunits the
  ? subunit, ß' subunit, ß subunit, ? subunit,
  and two a dimer subunits.
• however, the ? subunit is not a member of the
  core enzyme.

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The holoenzyme
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The core enzyme
b
a
b
a
w
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• Overall, the shape of RNA polymerase resembles
  a crab claw. The active site is found at the base
  of the pincers within a region called the active
  center cleft.

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Active center cleft
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• 2gt.The RNA product does not remain base-paired to
  the template DNA strand----rather, the enzyme
  displaces the growing chain only a few
  nucleotides behind where each ribonucleotide is
  added.

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• 3gt.Transcription, though very accurate, is less
  accurate than replication of DNA.
• This difference reflects the lack of extensive
  proofreading mechanisms for transcripts from a
  single gene in a short time.

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A series of steps for transcription

• Initiation
• Elongation
• Termination

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Initiation

• A promoter if the sequence that initially
  binds the RNA polymerase. As the replication, the
  DNA around the point where transcription will
  start unwinds, and the base pairs are disrupted.

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• Transcription always occurs in a 5 to 3
  direction.
• Only one DNA strand acts as the template
• on which RNA is built.
• The choice of promoter determines which
  stretch is transcribed at which regulation is
  imposed.

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Binding (closed complex)
Promoter melting (open complex)
Initial transcription
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Elongation

• Once the RNA polymerase has synthesized a short
  stretch of RNA (approximately ten bases), it
  shift into the elongation phase.
• This transition requires further comformational
• Changes in polymerase that lead it to grip the
• Template more firmly.

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Termination

• RNA polymerase stop and release the product
• In some cells, termination occurs at the specific
  and well-defined DNA sequences called
  terminators. Some cells lack such termination
  sequences.

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Three defined step of initiation

• Closed complex
• Open complex
• Stable ternary complex

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Closed complex

• The initial polymerase binding to the promoter
• DNA remains double stranded
• The enzyme is bound to one face of the helix

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Open complex

• the DNA strand separate over a distance of 14 bp
  (-11 to 3 ) around the start site (1 site)
• Replication bubble forms

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Stable ternary complex

• The enzyme escapes from the promoter
• The transition to the elongation phase
• Stable ternary complex
• DNA RNA enzyme

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Bacterial promoter

• s70 is the factor of E.coli RNA polymerase that
  has two conserved sequences, which are called
  consensus sequence. They are called -35 and -10
  region.
• Very few pomoters have this exact sequence, but
  most differ form it only by a few nucleotides.

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• Another class of s70 promoter lacks a 35 region
  and has an extended 10 element compensating
  for the absence of 35 region

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The s70 factor comprises four regions called s
region 1 to s region 4.

• Region 4 recognizes -35 element
• Region 2 recognizes -10 element
• Region 3 recognizes the extended 10 element

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• One helix inserts into the DNA major groove
  interacting with the bases at the 35 region. The
  other helix lies across the top of the groove,
  contacting the DNA backbone

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Interaction with 10 is more elaborate (??) and
less understood

• The -10 region is within DNA melting region
• The a helix recognizing 10 can interacts with
  bases on the non-template strand to stabilize the
  melted DNA

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UP-element is recognized by a carboxyl terminal
domain of the a-subunit (aCTD), but not by s
factor
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Transition to the open complex

• Structure changes open the DNA double helix to
  reveal the template and nontemplate strands. This
  melting occurs between positions -11 and 3, in
  relation to the transcription start site

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• For s70 containing RNA polymerase, isomerization
  is a spontaneous conformational change in the
  DNA-enzyme complex to a more energetically
  favorable form. (No extra energy requirement)

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The striking structural change in the polymerase

• 1. the b and b pincers down tightly on the
  downstream DNA
• 2. A major shift occurs in the N-terminal region
  of s (region 1.1) shifts. In the closed complex,
  s region 1.1 is in the active center in the open
  complex, the region 1.1 shift to the outside of
  the center, allowing DNA access to the cleft

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Transcription needs

• The initiating NTP (usually an A) is placed in
  the active site
• The initiating ATP is held tightly in the correct
  orientation by extensive interactions with the
  holoenzyme

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The elongating polymerase is a processive machine
that synthesizes and proofreads RNA
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Synthesizing by RNA polymerase

• DNA enters the polymerase between the pincers
• Strand separation in the catalytic cleft
• NTP addition
• RNA product spooling out (Only 8-9 nts of the
  growing RNA remain base-paired with the DNA
  template at any given time)
• DNA strand annealing in behind

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Proofreading by RNA polymerase

• Pyrohosphorolytic (?????)editing the enzyme
  catalyzes the removal of an incorrectly inserted
  ribonucleotide by reincorporation of PPi.
• Hydrolytic (??)editing the enzyme backtracks by
  one or more nucleotides and removes the
  error-containing sequence. This is stimulated by
  Gre factor, a elongation stimulation factor

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Transcription is terminated by signals within the
RNA sequence

• Terminator are the sequences that trigger the
  elongation polymerase to dissociate from the DNA
• There are two type of terminator
• Rho-independent and Rho-dependent

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Rho-independent terminator

• contains a short inverted repeat (20 bp) and a
  stretch of 8 AT base pairs

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Weakest base pairing AU make the dissociation
easier
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Rho -dependent terminators

• Have less well-characterized RNA elements, and
  requires Rho protein for termination
• Rho is a ring-shaped single-stranded RNA binding
  protein, like SSB
• Rho binding can wrest the RNA from the
  polymerase-template complex using the energy from
  ATP hydrolysis
• Rho binds to rut (r utilization) RNA sites
• Rho does not bind the translating RNA

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Transcription in eukaryotes

• Eukaryotes have different RNA polymerases while
  bacteria have only one.
• Several initiation factors ate required for
  efficient and promoter-specific initiation. These
  are called general transcription factors (GTFs)

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RNA polymerase II core promoters are made up of
combinations of 4 different sequence elements

• Eukaryotic core promoter (40 nt) the minimal
  set of sequence elements required for accurate
  transcription initiation by the Pol II machinery
  in vitro

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• TFIIB recognition element (BRE)
• The TATA element/box
• Initiator (Inr)
• The downstream promoter element (DPE)

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RNA polymerase form a pre-initiation complex with
general transcription factor at the promoter
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• TBP in TFIID binds to the TATA box
• TFIIA and TFIIB are recruited with TFIIB binding
  to the BRE
• RNA Pol II-TFIIF complex is then recruited
• TFIIE and TFIIH then bind upstream of Pol II to
  form the pre-initiation complex
• Promoter melting using energy from ATP hydrolysis
  by TFIIH )
• Promoter escapes after the phosphorylation of the
  CTD tail

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TBP binds to and distorts DNA using a sheet
inserted into the minor groove

• AT base pairs (TATA box) are favored because
  they are more readily distorted to allow initial
  opening of the minor groove

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The other GTFs also have specific roles in
initiation

• 10 TAFs (1) two of them bind DNA elements at
  the promoter (Inr and DPE) (2) several are
  histone-like TAFs and might bind to DNA similar
  to that histone does (3) one regulates the
  binding of TBP to DNA

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• TFIIB (1) a single polypeptide chain, (2)
  asymmetric binding to TBP and the promoter DNA
  (BRE), (3)bridging TBP and the polymerase, (4)
  the N-terminal inserting in the RNA exit channel
  resembles the s3.2 .

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• TFIIF (1) a two subunit factor, (2) binding of
  Pol II-TFIIF stabilizes the DNA-TBP-TFIIB
  complex, which is required for the followed
  factor binding
• TFIIE recruits and regulates TFIIH
• TFIIH (1) controls the ATP-dependent transition
  of the pre-initiation complex to the open
  complex, (2) contains 9 subunits and is the
  largest GTF two functions as ATPase and one is
  protein kinase. (3) important for promoter
  melting and escape. (4) ATPase functions in
  nucleotide mismatch repair, called
  transcription-coupled repair.

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in vivo, transcription initiation requires
additional proteins including the mediator complex
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Mediator

• includes more than 20 subunits
• Organized in modules

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A new set of factors stimulate pol elongation and
proofreading

• Transition from the initiation to elongation
  involves the Pol II enzyme shedding most of its
  initiation factors (GTF and mediators) and
  recruiting other factors

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• (1) Elongation factors factors that stimulate
  elongation, such as TFIIS and hSPT5.
• (2) RNA processing (RNA ??) factors
• Recruited to the C-terminal tail of the CTD of
  RNAP II to phosphorylate the tail for elongation
  stimulation, proofreading, and RNA processing
  like splicing and polyadenylation.

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elongation factors

• P-TEFb
• phosphorylates CTD
• Activates hSPT5
• Activates TAT-SF1
• TFIIS
• Stimulates the overall rate of elongation by
  resolving the polymerase pausing
• Proofreading

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Elongation polymerase is associated with a new
set of protein factors

• RNA processing
• Capping of the 5 end of the RNA
• Splicing of the introns (most complicated)
• Poly adenylation of the 3 end

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Function of poly(A) tail

• Increased mRNA stability
• Increased translational efficiency
• Splicing of last intron

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Function of 5cap

• Protection from degradation
• Increased translational efficiency
• Transport to cytoplasm
• Splicing of first intron

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Other processes

• Splicing joining the protein coding sequences
• 3 end polyadenylation

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• RNA Pol I III recognize distinct promoters ,
  using distinct sets of transcription factors, but
  still require TBP

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Thats all, thank you!