Mechanisms of Transcription

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Chapter 12

• Mechanisms of Transcription

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The Central Dogma

• DNA RNA Protein

transcription
translation
replication
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• We have discussed about the
  maintenance of the genome , which is the
  replication of the DNA. then it comes to the
  gene expression.

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• Gene expression is the process by which
  the information in the DNA double helix is
  converted into the RNA and proteins.
• and transcription is the first step involves
  copying DNA into RNA.

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• Compared with replication, transcription
  has something in common, that is
• DNA template is needed to synthesized a new
  chain of nucleotide.
• the direction is from 5' to 3'.

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• Also there several important differences
• only some regions of the genome are transcribed
  instead of the entire genome.
• the nucleotides used to build a new chain
• is ribonucleotides instead of
  deoxyribonucleotides.
• transcription does not require a primer.

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• The RNA product dose not remain base-paired to
  the DNA templates.
• less accurate than replication.

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Fig 1 transcription
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• RNA Polymerases and the
  Transcription cycle

Topic 1
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RNA Polymerases

• RNA polymerases performs the same reaction in
  all cells, they are highly conceived , thus the
  enzymes from these organisms share many features.

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• Bacteria have just one polymerase, as in
  E.coli, the basic enzyme called the core enzyme
  has 5 subunits one copy of each three
  subunits--- ß, ß ,and ?. and 2 copies of a.

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Fig 2 the crystal structure of prokaryotic
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• In eukaryotic cells, there are three
  polymerase, RNA Pol ?, ?, and ?. Pol ?and ? are
  involved in transcribing specialized,
  RNA-encoding genes. the former is for large
  ribosomal RNA precursor gene, the latter is for
  some nuclear RNA gene and the 5S rRNA.

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RNA Pol ? is the enzyme we will focus on, for it
is the most studied of the three.
Active center cleft
Fig 3 the crystal structure of eukaryotic
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Table 1 the subunits of RNA Polymerase
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• There are 5 channels ,each allows double-stranded
  DNA, template DNA, non-template DNA, rNTP, and
  RNA products into or out of the enzymes active
  center cleft.

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Transcription by RNA Polymerase Proceeds in a
Series of Steps

• Initiation
• Elongation
• Termination

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Fig 4 initiation of transcription
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initiation

• Promoter the DNA sequence which initially binds
  the RNA polymerase.
• The promoter-polymerase complex undergoes
  structure changes to proceed transcription.
• DNA at transcription site unwinds and forms a
  bubble.
• 3-end growing.

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Fig 5 elongation and termination of
transcription
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Elongation

• Once a short stretch of RNA about 10 bases
  synthesized, transcription turns into elongation
  phase.
• Further conformational change in polymerase
  required for gripping the template more firmly.
• functions synthesis RNA, unwinds the DNA and
  reseals behind, dissociates the growing RNA chain
  from template, proofreads.

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Termination

• Stop and release the RNA products.
• In some cells, specific and well-characterized
  sequences trigger termination, in others there
  are no such sequence.

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three defined steps in initiation

• Form closed complex.
• polymerase binds to a promoter, DNA helix
  remains double-stranded.
• Form open complex.
• DNA strands separate and form the
  transcription bubble

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• Promoter escape
• the transition to the elongation phase.
• form stable ternary complex. (containing
  polymerase, DNA, and RNA)

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Topic 2

• the Transcription Cycle in Bacteria

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• Bacterial promoters vary in strength and
  sequence, but have certain defining features.

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• the bacterial core RNA polymerase can initiate
  transcription at any point of a DNA molecule.
• However, in cells, the transcription only start
  at promoters. The point is the d factor.

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d factor
Core enzyme
Fig 6 RNA polymerase holoenzyme
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• It is the d factor that converts core enzyme into
  the form that initiates only at promoters.

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• Promoters share the characteristic structure
• Two conserved sequence, each of six nucleotides.
• Nonspecific stretch. 1719 nucleotides.

Fig 7 features of bacterial promoters
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The d factor has two conserved sequence that
recognize the promoters(-35 and -10 elements).
Between them is a non-specific stretch about 17bp.
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consensus sequence
Fig 8 regions of d
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• The sequence of -35 and -10 elements are not
  identical. The closer to the consensus sequence,
  the stronger the transcription will initiate.
• By the strength of a promoter, we mean how many
  transcripts it initiated in a given time.
• An Up-element is found in some strong promoters,
  which increases the polymerase binding by
  providing an additional interaction site.

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• The d factor mediates binding of polymerase to
  the promoter

As shown in fig 8, the d factor can
be divided into 4 regions Region 2---
recognize the -10 element Region 4--- recognize
the -35 element Region 3--- recognize the
extended -10 element
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• Two helices with the region 4 form a common
  DNA-binding motif called a helix-turn-helix
• ?One helix inserts into the major groove
  and interacts with bases in the -35 region.
• ?The other lies across the top of the
  groove, making contact with the DNA backbone.

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• The -10 region is recognized by an a helix. this
  interaction is less well-characterized and is
  more complicated
• ?within the element, DNA melting is
  initiated from the closed to open complex.
• ?the helix has to interact with bases on
  the nontemplate strand in a manner that
  stabilizes the melted DNA.

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• The UP-element is recognized by a carboxyl
  terminal domain of the a subunit, called the aCTD.

FIG 9 d and a subunits recruit RNA core enzyme
to the promoter
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• Transition from the closed complex to the open
  complex.
• This process is called isomerization. Which
  involves structure change of the enzyme and the
  opening of the DNA double helix.
• It is a spontaneous conformational change , as a
  result, it need not energy from ATP hydrolysis
  and is irreversible .

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NTP entering channel is back
DNA entering channel
Fig 10 channels into and out of the open complex
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• Two striking structure changes
• the ß and the ß pincers clamp down
  tightly on the downstream DNA.
• a major shift of the d region 1.1---when
  not bound to DNA, it blocks the pathway. In the
  open complex, it shift an angle out of the
  enzyme.

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• There is no need of primer for transcription
  because RNA polymerase can initiate an new RNA
  chain on template without a primer . In stead ,
  the initiating ATP is held tightly in the
  correct orientation by extensive interaction
  with the holoenzyme.

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RNA polymerase synthesis several short RNAs
before entering the elongation phase

• RNA synthesis begins
• an RNA an RNA
• less than 10 bp longer than 10bp
• Abortive initiation elongation phase

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• Abortive initiation . the transcripts are
  released from the polymerase, and the enzyme,
  without leaving the template, begins synthesis
  again

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• Structure barrier for the abortive initiation.
• the d region 3.2 lies in the middle of the
  RNA exit channel in the open complex.
• ejection of this region from the channel is
  necessary for elongation, and also takes the
  enzyme several attemps.

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The elongation polymerase is a processive machine
that synthesizes and proofreads RNA.

• Two proofreading functions
• Pyrophosphorolytic editing remove the incorrect
  ribonucleotide by reincorporation of PPi.
• Hydrolytic editing stimulated by Gre factors
  (the elongation factor ), draw back one or more
  ribonucleotides and cleaves them.

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Transcription is terminated by the signals within
the RNA sequence

• Terminator the sequence trigger the elongation
  polymerase to dissociate from the DNA and release
  the RNA products.

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• Rho-independent intrinsic terminator, requires
  no factor.
• Rho-dependent requires the Rho factor, and need
  ATP to wrest RNA from DNA and enzyme.

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a stretch of 8 A-T base
Fig 11 sequence of a rho-independent terminator
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• The hairpin cause the termination by disrupting
  the elongation complex. This is achieved either
  by forcing open the RNA exit channel in
  polymerase, or, by disrupting RNA-template
  interaction.

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Week base pairing---easy to dissociate
Fig 12 transcription termination
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• Rho-dependent terminators
• have less well-characterized RNA
  elements, and require Rho protein
• the protein is a ring-shaped
  single-stranded RNA binding protein, like SSB.
• wrest the RNA from the complex with ATP.
• Rho binds to rut RNA sites.

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Fig 13 the d transcription terminator
RNA tread through the ring
It has 6 subunits and also an ATPase activity
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Topic 3 transcription in eukaryotes
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• Transcription in eukaryotes is undertaken by
  polymerase closely related to the one in
  bacteria, but still there are some differences
• 3 polymerase (Pol? Pol? Pol ?) are found in
  eukaryotes while there is only one RNA polymerase
  in bacteria.
• Eukaryotes need several initiation factors
  (GFTs), bacteria only need one (d factor).

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• For DNA is packed into nucleosomes in vivo,
  several other factors besides GFTs are required
  in transcription .
• Mediator complex
• DNA-binding regulatory proteins
• Chromatin-modifying enzymes

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RNA polymerase ? core promoters are made up of
combinations of four different sequence elements

• A core promoter is typically about 40 nucleotides
  long, and the four sequence elements are
• BRE , TATA element , Inr , DPE

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TF?B recognition element
Initiator element
Downstream promoter element
TATA box
Fig 14 Pol ? core promoter
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• Typically there are only two or three of the four
  elements are included in promoter.
• Some other elements are required for efficient
  transcription, all these constitute the
  regulatory sequences (activators or repressors) .

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

• Pre-initiation complex GTFs Pol ? promoter
• TF?D TBP TAFs

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• The function of GTFs
• Help the enzyme binds the promoter
• Melt the DNA double helix.
• Help escaping from the promoter
• Embark on the elongation phase

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Binds the TATA element
Promoter melting requires ATP and is mediated by
TF?H
Fig 15 transcription initiation RNA pol ?
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Promoter escape

• Pol ? has a C- terminal domain (CDT), which
  extended as a tail.
• It contains a serious of repeats of the
  heptapeptide sequence, containing sites for
  phosphorylation.
• Those phosphates helps enzyme shed most of the
  GTFs, then it escapes from the promoter .

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

• TBP using a ß sheet to recognized the minor
  groove of the TATA element.
• ß sheet select the TATA element by structure
  distortion instead of chemical information.
• It favors the TATA element because the A-T bases
  are readily distorted.

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TBP
DNA
Fig 16 TBP-DNA complex
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The other GTFs also have specific roles in
initiation

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

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

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• TFIIF
• a two subunit factor
• binding of Pol II-TFIIF stabilizes the
  DNA-TBP-TFIIB complex, which is required for the
  followed factor binding
• TFIIE
• recruits and regulates TFIIH

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• TFIIH
• controls the ATP-dependent transition of the
  pre-initiation complex to the open complex
• contains 9 subunits and is the largest GTF two
  functions as ATPase and one is protein kinase.
• important for promoter melting and escape.
• ATPase functions in nucleotide mismatch repair,
  called transcription-coupled repair.

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in vivo, transcription initiation requires
additional proteins

• As it was said before, the DNA template in vivo
  is packed into nucleosome and chromatin, so
  additional proteins are needed

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• The mediator complex
• Transcriptional regulatory proteins
• Nucleosome-modifying enzymes

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Fig 17 assembly of the pre-initiation complex in
presence of mediator, nucleosome modifiers and
remodelers, and transcriptional activators
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Mediator consists of many subunits, some
conserved from yeast to human

• More than 20 subunits
• 7 subunits show significant sequence homology
  between yeast and human
• Only the subunit Srb4 is essential for
  transcription of essentially all Pol II genes in
  vivo
• Organized in modules

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Fig 18 comparison of the mediator
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A new set of factors stimulate Pol II elongation
and RNA proofreading

• Transition from the initiation to elongation
  involves the Pol II enzyme shedding most of its
  initiation factors (GTF and mediators)
• and then recruiting Elongation factors and RNA
  processing factors.

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• Elongation factors
• factors that stimulate elongation, such as
  TFIIS and hSPT5
• RNA processing 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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Fig 18 RNA processing enzymes are recruited by
the tail of polymerase
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Elongation polymerase is associated with a new
set of protein factors required for various types
of RNA processing

• RNA processing
• Capping of the 5 end of the RNA
• Splicing
• Poly adenylation of the 3 end

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• there is an overlap in proteins involving in
  those events, so, we can say elongation,
  termination of transcription, and RNA processing
  are interconnected.
• For examples
• The elongation factor hSPT5 also recruits and
  stimulates the 5 capping enzyme
• The elongation factor TAT-SF1 recruits components
  for splicing

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• Function of 5cap
• Protect from degradation
• Increased translational efficiency
• Transport to cytoplasm
• Splicing of first intron

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Fig 19 the structure and formation of the 5RNA
cap
The cap a methylated guanine joined
to the RNA transcript by a unusual 5-5 linkage
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Splicing kick out the introns and join the exons

• Dephosphorylation of Ser5 within the CTD tail
  leads to dissociation of capping machinery
• Further phosphorylation of Ser2 recruits the
  splicing machinery

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Poly adenylation of the 3 end

• The CTD tail is involved in recruiting the
  enzymes necessary for polyadenylation.
• The transcribed poly-A sequence triggers these
  events
• Cleavage of the message
• Addition of poly-A to its 3 end
• Termination of transcription

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Fig 20 polyadenylation and termination
ATP is needed as substrates
The addition of A requires no template
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• Two models are proposed to explain the linkage
  between polyadenylation and termination
• Model 1 The transfer of the 3 processing enzyme
  from CDT tail triggers conformational change that
  reduces processivity of the enzyme leading to
  spontaneous termination
• Model 2 absence of a 5cap on the second RNA
  molecule is sensed by the polymerase as improper
  and then terminate transcription

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RNA Pol I and III recognize distinct promoters ,
using distinct sets of transcription factors, but
still require TBP
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• Pol I transcribes rRNA precursor encoding gene ,
  which has many copies in a cell.
• Pol III transcribes tRNA genes, snRNA genes and
  5S rRNA genes
• The vast majority of Pol III have
  the unusual feature that the promoter is located
  downstream of the transcription start site.

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Fig 21 Pol I promoter region
Upstream control element
Fig 22 Pol III core promoter
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The end
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