Mechanisms of Transcription

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Mechanisms of Transcription
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• RNA polymerase does not need a primer to initiate
  transcription.
• The RNA product does not remain base-paired to
  the template DNA strand.
• Transcription is less accurate than replication.

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RNA polymerases and the transcription cycle

• RNA polymerase performs essentially the same
  reaction in all cells.
• From bacteria to mammals, the cellular RNA
  polymerases are made up of multiple subunits.

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Transcription is catalyzed by RNA polymerase,
which catalyzes the synthesis of RNA using DNA as
a template. RNA polymerase from E.coli contains
two ? subunits, ?, ?, ? and ?. The E. coli
holoenzyme is composed of a core, which is
competent to carry out RNA synthesis, and a s
factor, which directs the core to transcribe
specific genes.
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Comparison of the crystal structures of
prokaryotic and eukaryotic RNA polymerases
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Transcription in Prokaryotic cells Transcription
involves three stages
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• Initiation A promoter is the DNA sequence that
  initially binds the RNA polymerase. Only one of
  the DNA strands acts as a template. The choice of
  promoter determines which stretch of DNA is
  transcribed and is the main step at which
  regulation is imposed.

• Elongation Once the RNA polymerase has
  synthesized a short stretch of RNA approximately
  ten bases), it shifts into the elongation phase.

• Termination Once the polymerase has transcribed
  the length of the gene, it must stop and release
  the RNA product.

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The transcription cycle in bacteria

• Bacterial promoters vary in strength and
  sequence, but have certain defining features
• 3 regions of conservation -35, -10 and the
  length of spacer 17 - 19 bp, called consensus
  sequence.
• Promoters with sequences closer to the consensus
  sequence are generally stronger than those that
  match less well.

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• An additional DNA element, UP-element, that binds
  RNA polymerase is found in some strong promoters
  and increase polymerase binding by providing an
  additional specific interaction between the
  enzyme and the DNA.
• Another class of promoters lacks a -35 region and
  instead has a so-called extended -10 element.

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The sigma factors mediates binding of polymerase
to the promoter
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The best evidence for the functions of these
regions shows that sub-regions 2.4 and 4.2 are
involved in promoter 10 box and 35 box
recognition. The extended 10 element is
recognized by an ahelix in sregion 3.
s70 family There are four conserved regions in
sigma 70 family proteins.
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The RNA polymerase a subunit has an independently
folded C-terminal domain that can recognize and
bind to a promoters UP element. This allows very
tight binding between polymerase and
promoter. The a -CTD is connected to the aNTD by
a flexible linker. a subunit response to
activator, repressor, elongation factor and
transcription factors
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Transcription Initiation involves three defined
steps

• The first step in RNA synthesis is the binding of
  RNA polymerase to a DNA promoter- to form what is
  called a closed complex.
• In the second step, the closed complex undergoes
  a transition to the open complex in which the DNA
  strands separate over a distance of some 14 bp
  around the start site.

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• Once an RNA polymerase molecule has bound to a
  promoter site and locally unwound the DNA double
  helix, initiation of RNA synthesis can take
  place.
• Once the enzyme gets further than 10 bp, it is
  said to have escaped the promoter. A stable
  ternary complex contains enzyme, DNA and RNA.
  This is a transition to elongation phase.

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Transition to the open complex involves
structural changes in RNA polymerase and in the
promoter DNA
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The transition from closed to open complex
involves structural changes in the enzyme and the
opening of the DNA double helix to reveal the
template and nontemplate strands. In bacterial
enzyme with s70, this transition called
isomerization, does not require energy from ATP
hydrolysis. The active site of the enzyme, which
is made up of regions from both the ß and ß
subunits, is at the base of the pincers within
the active center cleft.
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Transcription is initiated by RNA polymerase
without the need for a primer

• This requires that the initiating ribonucleotide
  be brought into the active site and held stably
  on the template while the next NTP is presented
  with correct geometry for the polymerization to
  occur.
• The enzyme has to make specific interactions with
  the initiating NTP.
• The interactions are specific for the nucleotide
  on A, and only chains initiated with A are held
  in a manner suitable for efficient initiation.

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RNA polymerase synthesizes several short RNAs
before entering the elongation phase

• Abortive initiation the enzyme synthesizes short
  RNA molecules of less than ten nucleotides and
  then released from the polymerase. And the enzyme
  begins RNA synthesis again.
• Once a polymerase manages to make an RNA longer
  than 10 bp, a stable ternary complex is formed.
  This is the start of the elongation phase.
• Region 3.2 of s factor may be involved, and it
  mimics RNA.

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The elongation polymerase is a processive machine
that synthesizes and proofreading RNA

• Double-stranded DNA enters the front of the
  enzyme between the pincers.
• At the opening of the catalytic cleft, the
  strands separate to follow different paths
  through the enzyme before exiting via their
  respective channels and reforming a double helix
  behind the elongation polymerase.

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RNA polymerase carries out two proofreading
functions

• Pyrophosphorolytic editing the enzyme uses its
  active site to catalyze the removal of an
  incorrectly inserted NTP.
• Hydrolytic editing the polymerase backtracks by
  one or more nucleotides and cleaves the RNA
  product, removing the error-containing sequence.
• Hydrolytic editing is stimulated by Gre factors,
  which also serves as elongation stimulating
  factors.

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Transcription is terminated by signals within the
RNA sequences

• In bacteria, terminators come in two types
  rho-independent and rho-dependent.

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• Rho-independent Terminators, also called
  intrinsic terminators, a short inverted repeats
  (about 20 nucleotides) followed by a string of
  about eight AT bp.

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Rho-dependent terminators

• Have less well-characterized RNA elements and
  requires the action of the rho factor.
• Rho is an RNA helicase, composed of 6 identical
  subunits, each subunit has an RNA binding domain
  and ATPase domain, requires ATP to function.
• Rho releases the RNA product from the DNA
  template.

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How is Rho directed to a particular RNA molecule?

• There is some specificity in the sites it binds
  (the rut sites, Rho utilization). Optimally these
  sites consist of stretches of about 40
  nucleotides that do not fold into a secondary
  structure they are also rich in C.
• Rho fails to bind any transcript that is being
  translated.

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Transcription in Eukaryotes

• Eukaryotic cells have three different polymerase
  (Pol I, II and III)
• Whereas bacteria require only one initiation
  factor, several initiation factors are required
  for efficient and promoter-specific initiation in
  eukaryotes. These are called the general
  transcription factors (GTFs).

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Three different RNA polymerases

• RNA polymerase I resides in the nucleolus and is
  responsible for synthesizing three of the four
  types of rRNA found in eukaryotic ribosomes (28S,
  18S,and 5.8 S rRNA).
• RNA polymerase II is found in the nucleoplasm and
  synthesizes precursors to mRNA, the class of RNA
  molecules that code for proteins.
• RNA polymerase III is also a nucleoplasmic
  enzyme, but it synthesizes a variety of small
  RNAs, including tRNA precursors and the smallest
  type of ribosomal RNA, 5S rRNA.

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Core promoter defined as the minimal set of DNA
sequences sufficient to direct the accurate
initiation of transcription by RNA
polymerase Four types of DNA sequences are
involved in core promoter function in RNA
polymerase II (1) a short initiator (Inr) (2) the
TATA box (3) the TFIIB recognition element (BRE)
(4) the down stream promoter element (DPE)
RNA polymerase II core promoters are made up of
combinations of four different sequence elements
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Regulatory sequences

• Beyond the core promoter, there are other
  sequence elements required for efficient
  transcription in vivo.
• Together these elements constitute the regulatory
  sequences promoter proximal elements, upstream
  activator sequences, enhancers, silencers,
  boundary elements and insulators. All these
  elements bind regulatory proteins.

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RNA polymerase II forms a pre-initiation complex
with general transcription factors at the
promoter

• The complete set of general transcription factors
  and polymerase, bound together at the promoter
  and poised for initiation, is called the
  pre-initiation complex.

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In vitro, TFIIA, TFIIB, TFIIF together with
polymerase, and then TFIIE and TFIIH form the
pre-initiation complex. Promoter melting in
eukaryotes requires hydrolysis of ATP and is
mediated by TFIIH.
In eukaryotes, promoter escape involves
phosphorylation of the polymerase.
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• The largest subunit of Pol II has a C-terminal
  domain (CTD), which extends as a tail. The CTD
  contains a series of repeats of the heptapeptide
  sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. They are
  27 of these repeats in the yeast and 52 in the
  human case.
• Each repeat contains sites for phosphorylation by
  specific kinases including that is a subunit of
  TFIIH.

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TBP binds to and distorts DNA using a beta sheet
inserted into the minor groove
TBP uses an extensive region of ? sheet to
recognize the minor groove of the TATA
element. It also bends the DNA by an angle of
approximately 80o The interaction between TBP and
DNA involves only a limited number of hydrogen
bonds between the protein and the edges of the
base pairs in the minor groove.
TBP-DNA complex
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The other General Transcription factors also have
specific roles in initiation

• TAFs TBP is associated with about ten TAFs.
• TFIIB a single polypeptide chain, enters the
  pre-initiation complex after TBP. This protein
  appears to bridge the TATA-bound TBP and
  polymerase.
• TFIIF This two-subunit factor associated with
  PolII and is recruited to the promoter together
  with the enzyme.

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TFIIB-TBP-Promoter complex
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• TFIIE and TFIIHTFIIE recruits and regulates
  TFIIH.
• TFIIH controls the ATP-dependent transition of
  the pre-initiation complex to the open complex.
  It has nine subunits. Within TFIIH are two
  subunits that function as ATPases, and another
  that is a protein kinase, with roles in promoter
  melting and escape

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In vivo, transcription Initiation requires
additional Proteins, including the mediator
complex

• The high, regulated levels of transcription in
  vivo require the Mediator Complex,
  transcriptional regulatory proteins and in many
  cases, nucleosome-modifying enzymes.
• Mediator is associated with the CTD tail of the
  large polymerase subunit through one surface,
  while presenting other surfaces for interaction
  with DNA-bound activators.

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• Different Mediator subunits to bring polymerase
  to different genes.
• Mediator aids initiation by regulating the CTD
  kinase in TFIIH.
• The need for nucleosome modifiers and remodellers
  also differs at different promoters.

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Mediator consists of many subunits, some
conserved from yeast to human

• There are various forms of Mediator, each
  containing subsets of Mediator subunits.
• A complex consisting of Pol II, Mediator, and a
  some of the general transcription factors can be
  isolated from cells as a single complex in the
  absence of DNA---RNA Pol II holoenzyme.

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A new set of factors stimulate Pol II elongation
and RNA proofreading

• Elongation factors (such as TFIIS and hSPT5) are
  factors stimulate elongation.
• Phosphorylation of the CTD leads to an exchange
  of initiation factors for those factors required
  for elongation and RNA processing.
• Various proteins are thought to stimulate
  elongation by Pol II. One of them is the kinase
  P-TEFb.

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TFIIS

• TFIIS stimulates the overall rate of elongation
  by limiting the length of time polymerase pauses.
• TFIIS stimulates an inherent RNAse activity in
  polymerase and contributes to proofreading by
  polymerase.

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Kinase P-TEFb

• Kinase P-TEFb, is recruited to polymerase by
  transcriptional activators.
• Once bound to Pol II, it phosphorylates the
  serine residue at position 2 of the CTD.
• P-TEFb phosphorylates and activates hSPT5,
  another elongation factor. hSpT5 stimulates 5
  capping enzyme.
• P-TEFb also recruits TAT-SF1, an elongation
  factor, to stimulate elongation. TAT-SF1 recruits
  components of the splicing machinery.

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Elongating Polymerase is associated with a new
set of protein factors required for various types
of RNA Processing

• There is an overlap in proteins involved in
  elongation, and those required for RNA
  processing.
• hSPT5 recruits and stimulates the 5 capping
  enzyme. Elongation factor TAT-SF1 recruits
  components of the splicing machinery.

Phosphorylation on ser 5 capping
factors Phosphorylation on ser 2 splicing
factors
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The structure and formation of the 5 RNA cap

• The first RNA processing event is capping.
• It is a methylated guanine, and it is joined to
  the RNA transcript by an unusual 5 -5 linkage.
• The RNA is capped when it is still only 20-40
  nucleotides long- when the transcription cycles
  has progressed only to the transition between the
  initiation and elongation phases.

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Polyadenylation and termination

• The final RNA processing event, polyadenylation
  of the 3 end of the mRNA, is linked with the
  termination of trancription.
• The polymerase CTD tail is involved in recruiting
  the enzymes necessary for polyadenylation.
• Once polymerase has reached the end of a gene, it
  encounters specific sequences called poly-A
  signals.

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• CPSF (cleavage and polyadenylation specificity
  factor) and CstF (cleavage stimulation factor)
  are carried by the CTD of polymerase as it
  approaches the end of the gene.
• Once the CPSF and CstF are bound to the RNA,
  other proteins are recruited as well, leading
  initially to RNA cleavage and then
  polyadenylation.
• Polyadenylation signal is required for
  termination.

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

• Each of these enzymes also works with its own
  unique set of general transcription factors.
  However, TBP is universal for most of the cases.

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• RNA polymerase I resides in the nucleolus and is
  responsible for synthesizing three of the four
  types of rRNA found in eukaryotic ribosomes (28S,
  18S,and 5.8 S rRNA).
• The promoter for the rRNA genes comprise the
  core element and the UCE (upstream control
  element).
• Pol I promoter initiation requires Pol I, SL1 and
  UBF. SL1 comprises TBP and three TAFs, and binds
  DNA only in the presence of UBF.

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Pol III transcription

• Pol III initiation requires Pol III, TFIIIB and
  TFIIIC (for the tRNA genes), and those plus
  TFIIIA for the 5S rRNA gene.
• TFIIIC binds to the promoter region and recruits
  TFIIIB to the DNA just upstream of the start
  site, where it in turn recruits Pol III to the
  start site.