Welcome to Chapter 12

1
Welcome to Chapter 12

• Mechanisms of transcription

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Introduction

• Up to this point we have been considering
  maintenance to the genome ,that is ,how the
  genetic material is organized ,protected, and
  replicated.
• In the next parts ,we will describe the basic
  processed responsible for gene expression.
• First let us go into the world of transcription

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Transcription Vs Replication

• Transcription is chemically and enzymatically,
  very similar to DNA replication.Both involve
  enzymes that synthesize a new strand of nucleic
  acid complementary to DNA template
  strand.Moreover ,there are many differences
  between them.

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The differences go as follows

• RNA is made of ribonucleotides
• RNA polymerase ,which catalyzes the
  reaction,needs no primer
• The newly synthesized RNA does not remain
  base-paired to the template DNA strand
• Less accurate ,one mistake occurs in 10,000
• Because of different purpose ,transcription
  selectively copies only certain parts of the
  genome and makes anything from one to several
  hundred,or even thousand.

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Question why transcription is less accurate than
replication?

• I think the difference makes good sense if we
  associate it with the results of the mistakes.
• DNA is the molecule in which the genetic material
  is stored,and DNA replication si the process by
  which that genetic material is passed on.Any
  mistake can easily be catastrophicit becomes
  permanent in the genome of that individual and
  also gets passed on to subsequent generations.

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• Transcription ,in contrast,produces only
  transient copies and normally several from each
  transcribed region.
• Thus ,a mistake during transcription will rarely
  do more harm than render one out of many
  transient transcripts defective.

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Outline

• 1. RNA polymerase Transcription cycle
• 2. The transcription cycle in bacteria
• 3.Transcription in eukaryotes

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Topic 1

• RNA polymerase The transcription cycle

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RNA polymerase

• RNA pol come in different forms ,but share many
  features,especially in those parts of the enzyme
  directly involved with catalyzing the synthesis
  of RNA
• RNA pol performs essentially the same reaction in
  all cells,from bacteria to humans.

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1 The structure of RNA pol

• From bacteria to mammals ,the cellular RNA
  polymerase are made up of multiple subunits .
• Bacteria have only a single RNA pol ,which is the
  core enzyme capable of synthesizing RNA
• Eukaryotic cells have three, namely RNA pol I ,II
  ,and III .They are responsible for synthesis of
  different kinds of RNA

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Table 12-1 The subunits of RNA polymerases
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Crab claw shape of RNAP (The shape of DNA pol
is__)
Active center cleft
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b
Fig 12-2 RNAP Comparison
prokaryotic
a
b
The same color indicate the homologous of the two
enzymes
a
w
eukaryotic
RPB2
RPB3
RPB1
RPB11
RPB6
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• RNA pol II is the focus ,which is responsible for
  transcribing most genes-indeed,essentially all
  protein-encoding genes.
• RNA Pol I transcribes the large ribosomal RNA
  precursor gene.
• RNA Pol III transcribes tRNA genes,some small
  nuclear RNA genes,and the 5S rRNA gene

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• Since the structure of RNA Pol is this,there come
  the questionHow do they function? Or how do they
  realize the process of transcription?

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Transcription by RNA Pol proceeds in a series of
steps

• Initiation
• Elongation
• Termination
• Let us go deep into the details

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Process 1 Initiation

• (1)Promoter the DNA sequence that initially
  binds the RNA pol
• (2)Promoter-polymerase complex undergoes
  structural changes
• (3)The DNA around the point where transcription
  unwinds,forming a bubble( similar to DNA
  replication)
• (4)Again like DNA replication,the direction of
  transcription is from 5 to 3

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• Additionally ,unlike replication,only one of the
  DNA strands acts as a template on which the RNA
  strand is built.

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Transcription Initiation Invoves 3 Defined Steps

• Form closed complex
• Form open complex
• Form stable ternaty complex

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Fig 12-3-initiation
Binding (closed complex)
Promoter melting (open complex)
Initial transcription
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Closed complex

• Initial binding of pol to a promoter
• In this form ,DNA remains double-stranded,and the
  enzyme is bound to one face of the helix.

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Open complex

• DNA strands separate around the transcription
  site
• The transcription bubble forms

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Stable ternary complex

• Enzyme escape the promoter once it gets further
  than the 10 bp
• Stable ternary complex contains enzyme,DNA and
  RNA
• Then the elongation phase comes

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Process 2 Elongation

• Begins when the enzyme has synthesized a short
  stretch of RNA (about 10 bp)
• The RNA pol undergoes further comformational
  changes to grip the template more firmly
• The enzyme functionsRNA synthesis ,unwind the
  DNA chains in front,re-anneal it
  behind,dissociate the growing RNA chain from the
  template

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Fig 12-3-Elongation and termination
Elongation
Termination
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Process 3 Termination

• Once the length of the gene has been transcribed
  ,the RNA pol must stop and release the product
• In some cells ,there are specific,well-characteriz
  ed sequences.In other cells,it remains to be seen
  what instructs the termination

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Topic 2 The Transcription Cycle In Bacteria
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2-1 Bacterial promoters vary in strength
sequence,but have certain defining features

• The bacterial core RNA pol can ,in principle
  ,initiate transcription at any point on a DNA
  molecule .In cells,polymerase initiates
  transcription only at promoters.
• It is the addition of initiation factor called
  sthat converts core enzyme into the form that
  initiates only at promoters.
• That form of the enzyme is called holoenzyme
  ,which is made up of core enzyme and sfactor

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Fig 12-5a bacterial promoter
The distance is conserved

1. s70 promoters contain recognizable 35 and 10
   regions, but the sequences are not identical.
2. Comparison of many different promoters derives
   the consensus sequences reflecting preferred 10
   and 35 regions

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The details of s factor

• Structure composed of 4 regions called sregion
  1 through sregion 4
• Function recognize the site of promoter,
  mediates binding of polymerase to the promoter

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Fig 12-6 regions of s
Region 4 recognizes -35 element Region 2
recognizes -10 element Region 3 recognizes the
extended 10 element
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Figure 12-4

• Holoenzyme
• factor core enzyme

In cell, RNA polymerase initiates transcription
only at promoters. Who confers the polymerase
binding specificity?
,
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Take E.coli as a example

• In the case of E.coli ,the predominant sfactor
  is calleds70 factor .
• Promoters recognized by s70 factor share the
  following characteristic structuretwo conserved
  sequences,each of six nucleotides,are separated
  by a nonspecific stretch of 17-19nucleotides.
• The two defined sequences are centered,respectivel
  y,at about 10 bp and at about 35 bp upstream of
  the site where RNA synthesis starts.
• The sequences are thus called the 35 and 10
  regions,or elements.
• Position 1is the transcription start site.

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Consensus sequence

• Although the vast majority of s70 promoters
  contain recognizable 35 and 10 regions,the
  sequences are not identical.
• Comparison of many different sequences reflecting
  preferred 10 and 35 regions
• Promoters with sequences closer to the consensus
  are generally stronger than those that match
  less well.
• By the strength of a promoter,we mean how many
  transcripts it initiates in a given time.

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BOX 12-1 Figure 1
Consensus sequence of the -35 and -10 region
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Up-element

• An additional DNA element that binds RNA
  polymerase is found in some strong promoters
• Up-element can increases polymerase binding by
  providing an additional specific interaction
  between the enzyme and DNA
• The magnificence is this another class of s70
  promoters lacks a 35region and instead gas a so
  called extended-10 element,which compensates
  for the absence of 35 region.

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UP-element is recognized by a carboxyl terminal
domain of the a-subunit (aCTD), but not by s
factor
Fig 12-7 s and a subunits recruit RNA pol core
enzyme to the promoter
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Fig 12-5c bacterial promoter
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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2-2 The features of transcription in bacteria

• 1.Transition to the open complex involves
  structural changes in RNA pol and in the promoter
  DNA (melting , isomerization, the active center
  cleft)
• 2.Transcription is initiated by RNA pol without
  the need for a primer
• 3.RNA pol synthesizes several short RNAs before
  entering the elongation phase. (Abortive
  initiation)

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• 4.The elongating pol is a processive machine that
  synthesizes and proofreads RNA.(pyrophosphorolytic
  editing hydrolytic editing)
• 5.transcription is terminated by signals within
  the TNA sequence (Rho-independent Vs
  Rho-dependent, intrinsic terminators.)

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Rho-independent terminator contains a short
inverted repeat (20 bp) and a stretch of 8 AT
base pairs.
Fig 12-9
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Fig 12-11 the r transcription terminator
RNA tread trough the ring
Hexamer, Open ring
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Topic 3 transcription in eukaryotes
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Transcription in bacteria Vs in eukaryotes

• Eukaruotes have three different pol (I,II,III),
  whereas bacteria have only one.
• Bacteria require only one additional initiation
  factor(sfactor ) , but several initiation factors
  are required for efficient and promoter-specific
  initiation in eukaryotes,which is called the
  general transcription factors(GTFs)

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The factors needed for transcription in vivo

• GTFs
• Polymerase
• Mediator complex
• DNA-binding regulatory proteins
• Chromatin-modifying enzymes

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• However ,in vitro, the general transcription
  factors are all that is required,together with
  pol II .
• One reason for the difference is that the DNA
  template in vivo is packaged into nucleosomes and
  chromatin .This condition complicates binding to
  the promoter of pol and its associated factors.

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Core promoter

• Core promoter refers to the minimal set of
  sequence elements required for accurate
  transcription initiation by the pol II machinery.
• A core promoter is typically about 40 nucleotides
  long, extending either upstream or downstream of
  the transcription start site.

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Fig 12-12 Pol II core promoter

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

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Fore elements in core promoter

• BRE the TFIIB recognition element
• The TATA element
• Inr the initiator
• DPE the downstream promoter
• Generally , a promoter includes only two or three
  of these four elements .

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• Regulatory sequences
• Beyond the core promoter, there are other
  sequence elements required for efficient
  transcription in vivo.These elements constitute
  the regulatory sequences.
• They can be grouped into varous categories,
  reflecting their location, and the organism in
  question ,as much as their function

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The regulatory sequences include

• Promoter proximal elements
• Upstream activator sequences (UASs)
• Enhancers
• A series of repressing elements called
  silencers,boundary elements ,insulators .
• All of them bind regulatory elements ,which help
  or hinder transcription .

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Details of GTFs

• They can help pol bind to the promoter and melt
  the DNA.
• Also help pol escape from the promoter and embark
  on the elongation phase.
• Pre-initiation complex GTFs promoter ,
• can initiate the transcription .

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Formation of pre-initiation complex

• TFIID recognizes the TATA element
• TBP formed when TFIID binds to the TATA element
• Another subunits on this complex are called TAFs
  ,for TBP associated factors .
• Other GTFs involved are TFIIA ,B, F,E, H

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Something about TBP

• TBP binds to and distorts DNA using a ßsheet
  inserted into the minor groove ,while typically
  proteins recognize DNA using ahelices inserted
  into the major groove of DNA.
• The reason for TBPs unorthodox recognition
  mechanism is linked to the need for that protein
  to distort the local DNA structure.

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TBP binds to and distorts DNA using a b sheet
inserted into the minor groove
The transcription in eukaryotes

• Unusual (P367 for the detailed mechanism)
• The need for that protein to distort the local
  DNA structure

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TBP binds to and distorts DNA using a b sheet
inserted into the minor groove
The transcription in eukaryotes

• Unusual (P367 for the detailed mechanism)
• The need for that protein to distort the local
  DNA structure

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The other GTFs also have specific roles in
initiation

• 1.TAFs
• Two of them bind DNA elements at the promoter
• several of them have structural homology to
  histone proteins
• Another appears to regulate the binding of TBP to
  DNA ,using an inhibitory
• 2.TFIIB
• This protein ,a single polypeptide chain,enter
  the pre-initiation complex after TBP

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• 3.TFIIE
• It has two subunits ,associating with pol II
  and recruited to the promoter together with that
  enzyme.
• 4.TFIIETFIIH
• TFIIE,which ,like TFIIF, consists of two
  subunits ,binds next and has roles in the
  recruitment and regulation of TFIIH,which
  controls the ATP-dependent transition of the
  pro-initiation complex to the open complex

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The C-terminal Domain

• The contraction is CTD
• In the shape of tail
• Containing a series of the heptapeptide sequence.
• Involved in the abortive initiation , promoter
  escape.
• Control later steps involving processing of the
  RNA

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Mediator complex

• Consists of many subunits (more than 20), some
  conserved from yeast to human .
• There are 7 subunits of 20 ones showing sequence
  homology between the two organisms.
• Few of them have any identified function.
• Only one is essential for transcription of
  essentially all pol II genes in vivo.

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Fig 12-17 comparison of the yeast and human
mediators
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Fig 12-16 assembly of the pre-initiation complex
in presence of mediator, nucleosome modifiers and
remodelers, and transcriptional activators
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RNA Pol II holoenzyme ?

• The dissociation arises the question that whether
  the RNA Pol II holoenzyme exists
• The enzyme is a complex consisting of pol
  II,Mediator, and some of the GTFs
• Sometimes ,the complex can be isolated from cells
  as a single one in the absence of DNA

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Elongation factors

• A new set of factors stimulate pol II
  elongation and RNA proofreading.
• (1)CTD
• The phosphorylation of the CTD leads to an
  exchange of initiation factors for those factors
  required for elongation and RNA processing.

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Beside CDT,various proteins are thought to
stimulate elongation by pol II

• The kinase P-TEFb recruited to polymerase by
  transcriptional activators.
• TAT-SF1 recruited by P-TEFb
• TEIIS does not affect initiation , but
  stimulates elongation contributes to
  proofreading by pol .

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RNA processing

• Elongating pol is associated with a new set of
  protein factors required for various types for
  RNA processing
• Once transcribed, eukaryotic RNA has to be
  processed in various ways before being exported
  from the nucleus where it can be translated.
• In fact ,elongation , termination of
  transcription,and RNA processing are
  interconnected-presumably to ensure their proper
  coordination

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Fig 12-18 RNA processing enzymes are recruited by
the tail of polymerase
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The processing events include

• Splicing the most complicated
• Capping of the 5 end of RNA the first RNA
  processing event ,involving the addition of a
  modified guanine base to the 5 end of the RNA .
• Polyadenylation of the 3 end of the RNA
  mediated by poly-A polymerase .

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RNA processing 15 end capping

• The cap a methylated guanine joined to the RNA
  transcript by a 5-5 linkage
• The linkage contains 3 phosphates
• 3 sequential enzymatic reactions
• Occurs early

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RNA processing 15 end capping

• The cap a methylated guanine joined to the RNA
  transcript by a 5-5 linkage
• The linkage contains 3 phosphates
• 3 sequential enzymatic reactions
• Occurs early

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Splicing joining the protein coding sequences

• 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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1. CPSF (cleavage and polyadenylation specificity
factor) CstF (cleavage stimulation factor) bind
to the poly-A signal, leading to the RNA cleavage
2. Poly-A polymerase (PAP) adds 200 As at the
3 end of the RNA, using ATP as a substrate
Fig 12-20 polyadenylation and termination
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RNA Pol IRNA Pol III

• RNA Pol I and III recognize distinct promoters
  ,using distinct sets of transcription factors
  ,but still require TBP
• Different from RNA Pol II, they transcribe
  distinct genes encoding specialized RNAs ,rather
  than proteins.

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RNA Pol I

• Requred for the expression of only one gene ,that
  encoding the rRNA precursor .
• The gene transcribed by RNA Pol I is expressed at
  a extremely high level.
• Comprises of two parts the core element the
  UCE
• Initiates with existence of SL1UBF

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Pol I promoter recognition
Upstream control element
UBF binds to the upstream of UCE, bring SL1 to
the downstream part of UCE. SL1 in turn recruits
RNAP I to the core promoter for transcription
Fig 12-21 Pol I promoter region
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RNA Pol III

• Pol III promoters come in various forms,and the
  vast majority have the unusual feature of being
  located downstream of the transcription start
  site.
• The factors required for transcription are called
  TFIIIB and TFIIIC ,and those plus TFIIIA for the
  5S rRNA gene.

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Pol III promoter recognition1. Different forms,
2. locates downstream of the transcription site
TFIIIC binds to the promoter, recruiting TFIIIB,
which in turn recruits RNAP III
Fig 12-22 Pol III core promoter
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SUMMARY

• RNA polymerase crab claw structure function
  (mediated the transcription)
• Transcription Vs replication
• Transcription cycle initiation ,elongation, and
  termination
• Transcription in bacteria (sfactor )
• Transcription in eukaryotes (RNA pol I , II ,III
  GTFs , core promoters regulatory sequence )

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