Molecular Biology Course

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• Molecular Biology Course

Section K Transcription in prokaryotes
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Section K Transcription in prokaryotes
K1 Basic principles of transcription
An overview, the process of RNA synthesis (
initiation, elongation, termination)
K2 Escherichia coli RNA polymerase
Properties, a subunit, b subunit, b subunit,
sigma (s) factor
K3 The E. coli s70 promoter
Promoter, s70 size, -10 sequence, -35 sequence,
transcription start site, promoter efficiency
K4 transcription process.
Promoter binding, unwinding, RNA chain
initiation, elongation, termination (r factor)
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• Molecular Biology Course

K1 Basic principles of transcription

• Transcription an overview (comparison with
  replication)
• The process of RNA synthesis initiation,
  elongation, termination

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• Molecular Biology Course

K1-1 Transcription an overview
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Key terms defined in this section (Gene VII)
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Gene X
upstream
downstream
Primary transcript
m7Gppp
mRNA
AAAAAn
Coding strand of DNA has the same sequence as
mRNA.Downstream identifies sequences proceeding
further in the direction of expression for
example, the coding region is downstream of the
initiation codon.
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Upstream identifies sequences proceeding in the
opposite direction from expression for example,
the bacterial promoter is upstream from the
transcription unit, the initiation codon is
upstream of the coding region. Transcription
unit is the distance between sites of initiation
and termination by RNA polymerase may include
more than one gene. Promoter is a region of DNA
involved in binding of RNA polymerase to initiate
transcription
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RNA Terminator is a sequence of DNA, represented
at the end of the transcript, that causes RNA
polymerase to terminate transcription. RNA
polymerases are enzymes that synthesize RNA using
a DNA template (formally described as
DNA-dependent RNA polymerases). Primary
transcript is the original unmodified RNA product
corresponding to a transcription unit.
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K1 Basic principles of transcription
Replication synthesis of two DNA molecules using
both parental DNA strands as templates.
Duplication of a DNA molecule. 1 DNA molecule ?
2 DNA molecules Transcription synthesis of one
RNA molecule using one of the two DNA strands as
a template. 1 DNA molecule ? 1 RNA molecule
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Review of replication
Replication-synthesis of the leading strand
the same direction as the replication fork moves
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Review of replication
Replication- Synthesis of the Okazaki fragments
Opposite to the replication fork movement
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Coupling the synthesis of leading and lagging
strands with a dimeric DNA pol III (E. coli)
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K1 Basic principles of transcription
Transcription
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K1 Basic principles of transcription

• RNA synthesis occurs in the 5?3 direction and
  its sequence corresponds to the sense strand
  (coding strand).
• The template of RNA synthesis is the antisense
  strand (template strand).
• Phosphodiester bonds same as in DNA
• Necessary components RNA polymerase,
  transcription factors, rNTPs, promoter
  terminator/template

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K1 Basic principles of transcription
K1-2 The process of RNA synthesis

• initiation
• elongation
• termination

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Flowchart of RNA synthesis
Back 1, 2
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K1 Basic principles of transcription
Fig. 2. Structure of a typical transcription unit
Is transcribed region equal to coding region? Why?
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K1 Basic principles of transcription
Initiation (template recognition)

• Binding of an RNA polymerase to the dsDNA
• Slide to find the promoter
• Unwind the DNA helix
• Synthesis of the RNA strand at the start site
  (initiation site), this position called position
  1

Link
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K1 Basic principles of transcription
Elongation

• Covalently adds ribonucleotides to the 3-end of
  the growing RNA chain.
• The RNA polymerase extend the growing RNA chain
  in the direction of 5? 3
• The enzyme itself moves in 3 to 5 along the
  antisense DNA strand.

Link
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K1 Basic principles of transcription
Termination

• Ending of RNA synthesis the dissociation of the
  RNA polymerase and RNA chain from the template
  DNA at the terminator site.
• Terminator often contains self-complementary
  regions which can form a stem-loop or hairpin
  structure in the RNA products (see K4 for details)

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Terminator structure
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K2 Escherichia coli RNA polymerase

• E. coli RNA polymerase
• a subunit
• b subunit
• b subunit
• sigma (s) factor

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K2 E. coli RNA polymerase
K2-1 E. coli RNA polymerase
Synthesis of single-stranded RNA from DNA
template.
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K2 E. coli RNA polymerase
RNA polymerase
(NMP)n NTP ? (NMP)n1 PPi

• Requires no primer for polymerization
• Requires DNA for activity and is most active with
  a double-stranded DNA as template.
• 5 ? 3 synthesis
• Require Mg2 for RNA synthesis activity
• lacks 3 ? 5 exonuclease activity, and the error
  rate of nucleotides incorporation is 10-4 to
  10-5. Is this accuracy good enough for gene
  expression??
• 6. usually are multisubunit enzyme.

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K2 E. coli RNA polymerase
E. coli polymerase

• E. coli has a single DNA-directed RNA polymerase
  that synthesizes all types of RNA.
• One of the largest enzyme in the cells
• Consists of at least 5 subunits in the
  holoenzyme, 2 alpha (a), and 1 of beta (b), beta
  prime (b), omega (w) and sigma (s) subunits
• Shaped as a cylindrical channel that can bind
  directly to 16 bp of DNA. The whole polymerase
  binds over 60 bp.
• RNA synthesis rate 40 nt per second at 37oC

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E. coli RNA polymerase
K2 E. coli RNA polymerase
155 KD
36.5 KD
11 KD
36.5 KD
70 KD
Initiation only
151 KD
Both initiation elongation
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K2 E. coli RNA polymerase

• The polymerases of bacteriophage T3 and T7 are
  smaller single polypeptide chains, they
  synthesize RNA rapidly (200 nt/sec) and recognize
  their own promoters which are different from E.
  coli promoters.

RNA polymerase differs from organism to organism
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K2 E. coli RNA polymerase

K2-2 a subunit
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E. coli polymerase a subunit
K2 E. coli RNA polymerase

• Two identical subunits in the core enzyme
• Encoded by the rpoA gene
• Required for assembly of the core enzyme
• Plays a role in promoter recognition. Experiment
  When phage T4 infects E. coli, the a subunit is
  modified by ADP-ribosylation of an arginine. The
  modification is associated with a reduced
  affinity for the promoters formerly recognized by
  the holoenzyme.
• plays a role in the interaction of RNA
  polymerase with some regulatory factors

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K2 E. coli RNA polymerase
K2-34 b and b subunit
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• b is encoded by rpoB gene, and b is encoded by
  rpoC gene .
• Make up the catalytic center of the RNA
  polymerase
• Their sequences are related to those of the
  largest subunits of eukaryotic RNA polymerases,
  suggesting that there are common features to the
  actions of all RNA polymerases.
• The b subunit can be crosslinked to the template
  DNA, the product RNA, and the substrate
  ribonucleotides mutations in rpoB affect all
  stages of transcription. Mutations in rpoC show
  that b also is involved at all stages.

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K2 E. coli RNA polymerase

• b subunit may contain two domains responsible for
  transcription initiation and elongation
• Rifampicin (???)has been shown to bind to the ß
  subunit, and inhibit transcription initiation by
  prokaryotic RNA pol. Mutation in rpoB gene can
  result in rifampicin resistance.
• Streptolydigins(?????)resistant mutations are
  mapped to rpoB gene as well. Inhibits
  transcription elongation but not initiation.

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K2 E. coli RNA polymerase

• b subunit
• Binds two Zn 2 ions and may participate in the
  catalytic function of the polymerase
• Hyparin (??)binds to the b subunit and inhibits
  transcription in vitro.
• Hyparin competes with DNA for binding to the
  polymerase.
• 2. b subunit may be responsible for binding to
  the template DNA .

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K2 E. coli RNA polymerase
K2-5 Sigma (s) factor
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• Many prokaryotes contain multiple s factors to
  recognize different promoters. The most common s
  factor in E. coli is s70.
• Binding of the s factor converts the core RNA pol
  into the holoenzyme.
• s factor is critical in promoter recognition, by
  decreasing the affinity of the core enzyme for
  non-specific DNA sites (104) and increasing the
  affinity for the corresponding promoter
• s factor is released from the RNA pol after
  initiation (RNA chain is 8-9 nt)
• Less amount of s factor is required in cells than
  that of the other subunits of the RNA pol.

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• Molecular Biology Course

K3 The E. coli s70 promoter

• Promoter
• s70 size
• -10 sequence
• -35 sequence
• transcription start site
• promoter efficiency

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K3 The E. coli s70 promoter
K3-1 Promoter

• The specific short conserved DNA sequences
• upstream from the transcribed sequence, and thus
  assigned a negative number (location)
• required for specific binding of RNA Pol. and
  transcription initiation (function)
• Were first characterized through mutations that
  enhance or diminish the rate of transcription of
  gene

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K3 The E. coli s70 promoter
Different promoters result in differing
efficiencies of transcription initiation, which
in turn regulate transcription.
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(No Transcript)
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K3 The E. coli s70 promoter
K3-2,34 s70 promoter
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K3 The E. coli s70 promoter
---5-8 bp---
G C T A
TTGACA
-----16-18 bp-------
TATAAT
-35 sequence
-10 sequence
1

• Consists of a sequence of between 40 and 60 bp
• -55 to 20 bound by the polymerase
• -20 to 20 tightly associated with the
  polymerase and protected from nuclease digestion
  by DNase?(see the supplemental)
• Up to position 40 critical for promoter
  function (mutagenesis analysis)
• -10 and 35 sequence 6 bp each, particularly
  important for promoter function in E. coli

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-10 sequence (Pribonow box)
K3 The E. coli s70 promoter

• The most conserved sequence in s70 promoters at
  which DNA unwinding is initiated by RNA Pol.
• A 6 bp sequence which is centered at around the
  10 position, and is found in the promoters of
  many different E. coli gene.
• The consensus sequence is TATAAT. The first two
  bases (TA) and the final T are most highly
  conserved.
• This hexamer is separated by between 5 and 8 bp
  from position 1, and the distance is critical.

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-35 sequence enhances recognition and
interaction with the polymerase s factor
K3 The E. coli s70 promoter

• A conserved hexamer sequence around position 35
• A consensus sequence of TTGACA
• The first three positions (TTG) are the most
  conserved among E. coli promoters.
• Separated by 16-18 bp from the 10 box in 90 of
  all promoters

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RNA Polymerase Leaves Its FootPrint on a Promoter
Supplemental material

• Footprinting is a technique derived from
  principles used in DNA sequencing. It is used to
  identify the specific DNA sequences that are
  bound by a particular protein.

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Supplemental material
Footprinting
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Supplemental material
Footprinting
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K3 The E. coli s70 promoter
K3-5 Transcription start site

• Is a purine in 90 of all gene
• G is more common at position 1 than A
• There are usually a C and T on either side of
  the start nucleotide (i.e. CGT or CAT)

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K3 The E. coli s70 promoter
The sequences of five E. coli promoters
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K3 The E. coli s70 promoter
K3-6 promoter efficiency
There is considerable variation in sequence
between different promoters, and the
transcription efficiency can vary by up to
1000-fold .
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• The 35 sequence constitutes a recognition region
  which enhances recognition and interaction with
  the polymerase s factor.
• The -10 sequence is important for DNA unwinding.
• The sequence around the start site influence
  initiation efficiency.
• The sequence of the first 30 bases to be
  transcribed controls the rate at which the RNA
  polymerase clears the promoter, hence influences
  the rate of the transcription and the overall
  promoter strength.

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Weak promoters and activating factor
Some promoter sequence are not sufficiently
similar to the consensus sequence to be strongly
transcribed under normal condition, thus
activating factor is required for efficient
initiation. Example Lac promoter P lac
requires activating protein, cAMP receptor
protein (CRP ), to bind to a site on the DNA
close to the promoter sequence in order to
enhance polymerase binding and transcription
initiation.
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• Molecular Biology Course

K4 Transcription process

• Promoter binding
• DNA unwinding
• RNA chain initiation
• RNA chain elongation
• RNA chain termination (r factor)

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K4 Transcription process

• Promoter binding

The searching process is extremely rapidly
Closed complex the initial complex of the
polymerase with the base-paired promoter DNA)
and 10 region
Link
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K4 Transcription process
The role of s factor in promoter binding

• The RNA polymerase core enyzme, a2bbw, has a
  general non-specific affinity for DNA, which is
  referred to as loose binding that is fairly
  stable.
• The addition of s factor to the core enzyme
  markedly reduces the holoenzyme affinity for
  non-specific binding by 20 000-fold, and enhances
  the holoenzyme binding to correct promoter sites
  100 times.
• Overall, s factor binding dramatically increases
  the specificity of the holoenzyme for correct
  promoter-binding site.

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K4 Transcription process
2. DNA unwinding
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The initial unwinding of the DNA results in
formation of an open complex with the polymerase,
and this process is referred to as tight binding
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K4 Transcription process
Negative supercoiling unwinding

• It is necessary to unwind the DNA so that the
  antisense strand to become accessible for base
  pairing and RNA synthesis.
• Negative supercoiling enhances the transcription
  of many genes, since it facilitates unwinding.
  Some promoters are not.
• Exceptional example promters for the enzyme
  subunits of DNA gyrase are inhibited by negative
  supercoiling, serving as an elegant feedback
  loop for DNA gyrase expression.

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K4 Transcription process
3. RNA chain initiation
Starts with a GTP or ATP
The polymerase initially incorporates the first
two nucleotides and forms a phosphodiester bond.
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Abortive initiation
K4 Transcription process
The first 9 nt are incorporated without
polymerase movement along the DNA. Afterward,
there is a significant probability that the chain
will be aborted.

• The RNA pol. goes through multiple abortive
  initiations before a successful initiation, which
  limits the overall rate of transcription
• The minimum time for promoter clearance is 1-2
  seconds (a long event, the synthesis is 40 nt/
  sec)

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K4 Transcription process
4. RNA chain elongation
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K4 Transcription process

• Promoter clearance when initiation succeeds, the
  enzyme releases s factor and forms a ternary
  complex of polymerase-DNA-nascent RNA, causing
  the polymerase to progress along the DNA to allow
  the re-initiation of transcription.

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K4 Transcription process

• Transcription bubble
• containing 17 bp of unwound DNA region and the
  3-end of the RNA that forms a hybrid helix about
  12 bp.
• moves along the DNA with RNA polymerase which
  unwinds DNA at the front and rewinds it at the
  rear.
• The E. coli polymerase moves at an average rate
  of 40 nt per sec, depending on the local DNA
  sequence.

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Transcription bubble
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5. RNA chain termination

• Termination occurs at terminator DNA sequences.
• The most common stop signal is an RNA hairpin
  (self-complement structure)
• commonly GC-rich to favor the structure
  stability
• 3. Rho-dependent and Rho-independent Termination.
  

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Terminator A specific DNA sequence where the
transcription complex dissociate

• Rho protein (r) independent terminator contains
• self-complementary region that is G-C rich and
  can form a stem-loop or hairpin secondary
  structure. GC-rich favouring the base pairing
  stability and causing the polymerase to pause.
• a run of adenylates (As) in the template strand
  that are transcribed into uridylates (Us) at the
  end of the RNA, resulting in weak RNA-antisense
  DNA strand binding.

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A model for r-independent termination of
transcription in E. coli.
The A-U base-pairing is less stable that favors
the dissociation
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Rho protein (r) dependent terminator

• Contains only the self-complementary region
• Requires r protein for termination
• r protein binds to specific sites in the
  single-stranded RNA
• r protein hydrolyzes ATP and moves along the
  nascent RNA towards the transcription complex
  then enables the polymerase to terminate
  transcription

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(No Transcript)
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RNA polymerase/transcription and DNA
polymerase/replication
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K supplemental 1
In any chromosome, different genes may use
different strands as template (Fig. 25-2).
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Thanks