Transcription

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Transcription

• Lecture 12
• Dr. Attya Bhatti

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Central dogma of molecular Biology
FigureThe three processes of information
transfer replication, transcription, and
translation.
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Properties of RNA

• RNA is usually single stranded, not a double
  helix.
• RNA has the sugar ribose in its nucleotides,
  rather than deoxyribose. The two sugars differ in
  the presence or absence of just one oxygen atom.
• RNA nucleotides carry the bases adenine, guanine,
  and cytosine, but the pyrimidine base uracil (U)
  is found in place of thymine. However, uracil
  does form hydrogen bonds with adenine, just as
  thymine does.

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Basic Requirements

• A template. The preferred template is
  double-stranded DNA. Single-stranded DNA also can
  serve as a template.
• RNA, whether single or double stranded, is not an
  effective template nor are RNA-DNA hybrids.
• Activated precursors. All four ribonucleoside
  triphosphates ATP, GTP, UTP, and CTP are
  required.
• A divalent metal ion. Mg2 or Mn2 are effective.

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Similarities in Replication and Transcription

• First, the direction of synthesis is 5 ?3 .
• Second, the mechanism of elongation is similar
  the 3 -OH group at the terminus of the growing
  chain makes a nucleophilic attack on the
  innermost phosphate of the incoming nucleoside
  triphosphate.
• Third, the synthesis is driven forward by the
  hydrolysis of pyrophosphate.

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• All three types of cellular RNA mRNA, tRNA, and
  rRNA are synthesized in E. coli by the same RNA
  polymerase according to instructions given by a
  DNA template.

• In mammalian cells, there is a division of labor
  among several different kinds of RNA polymerases.

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Transcription

• It occurs in three distinct stages
• Initiation,
• Elongation,
• and
• Termination.

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Initiation

• The regions of the DNA that signal initiation of
  transcription in prokaryotes are termed
  promoters.
• Promoter sites have regions of similar sequences.
  

Figure Promoter sequence. The promoter lies
upstream (toward 5' end) of the initiation
point and coding sequences.
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• The bases that appear just before the first base
  transcribed are designated the initiation site
  .
• Transcription relies on the complementary pairing
  of bases.
• The two strands of the double helix separate
  locally, and one of the separated strands acts as
  a template.
• Next, free nucleotides are aligned on the DNA
  template by their complementary bases in the
  template.
• The free ribonucleotide A aligns with T in the
  DNA, G with C, C with G, and U with A in RNA.

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RNA polymerase

• The process is catalyzed by the enzyme RNA
  polymerase .
• RNA growth is always in the 5'?3' direction in
  other words, nucleotides are always added at a 3'
  growing tip.
• Because of the antiparallel nature of the
  nucleotide pairing, the fact that RNA is
  synthesized 5'?3' means that the template strand
  must be oriented 3'?5'.
• In most prokaryotes, a single RNA polymerase
  species transcribes all types of RNA.

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Figure Initiation of transcription. (a) RNA
polymerase searches for a promoter site. (b) It
recognizes a promoter site and binds tightly,
forming a closed complex. (c) The holoenzyme
unwinds a short stretch of DNA, forming an open
complex. Transcription begins.
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• First, the holoenzyme (Polymerase) searches for a
  promoter (Figure 10-9a)
• and initially binds loosely to it, recognizing
  the -35 and -10 regions.
• The resulting structure is termed a closed
  promoter complex (Figure 10-9b).
• Then, the enzyme binds more tightly, unwinding
  bases near the -10 region. When the bound
  polymerase causes this local denaturation of the
  DNA duplex, it is said to form an open promoter
  complex (Figure 10-9c).
• This initiation step, the formation of an open
  complex, requires the sigma factor.

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Elongation

• Shortly after initiating transcription, the sigma
  factor dissociates from the RNA polymerase.
• The RNA is always synthesized in the 5'?3'
  direction with nucleoside triphosphates (NTPs)
  acting as substrates for the enzyme.

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Figure Transcription by RNA polymerase. An RNA
strand is synthesized in the 5'?3' direction from
a locally single stranded region of DNA.
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Termination

• There are two major mechanisms for termination in
  E. coli.
• 1st method (Direct method),
• The terminator sequences contain about 40 bp,
  ending in a GC-rich stretch that is followed by a
  run of six or more A's on the template strand.
• The corresponding GC sequences on the RNA are so
  arranged that the transcript in this region is
  able to form complementary bonds with itself,
  forming hairpin loop.
• It is followed by the terminal run of U's that
  correspond to the A residues on the DNA template.
  
• The hairpin loop and section of U residues appear
  to serve as a signal for the release of RNA
  polymerase and termination of transcription.

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Figure The structure of a termination site for
RNA polymerase in bacteria. The hairpin structure
forms by complementary base pairing within the
RNA strand.
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• In the second type, the help of an additional
  protein factor, termed rho, is required for RNA
  polymerase to recognize the termination signals.
• mRNAs with rho-dependent termination signals do
  not have the string of U residues at the end of
  the RNA and usually do not have hairpin loops.
• Rho is a hexamer consisting of six identical
  subunits the hydrolysis of ATP to ADP and Pi
  drives the termination reaction.
• The first step in termination is the binding of
  rho to a specific site on the RNA termed rut.

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• After binding, rho pulls the RNA off the RNA
  polymerase, probably by translocating along the
  mRNA.
• The rut sites are located just upstream from
  sequences at which the RNA polymerase tends to
  pause.
• The efficiency of both mechanisms of termination
  is influenced by surrounding sequences and other
  protein factors, as well.

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Fig A model for rho action on a nascent
cotranslated mRNA.