Chapter 12: Mechanisms of Transcription

1
Chapter 12 Mechanisms of Transcription

• ? RNA Polymerases and the Transcription Cycle
• ? The Transcription Cycle in Bacteria
• ? Transcription in Eukaryotes

2
Transcription is, chemically and enzymatically,
very similar to DNA replication. However, there
are some important differences.

• Differences
• 1.RNA is made from ribonucleotides.
• 2.RNA polymerase catalyzes the reaction.
• 3.RNA polymerase does not need a primer.
• 4.The RNA product does not remain base-paired to
  the template DNA strand. (Figure12-1)
• 5.Transcription is less accurate than
  replication.
• 6. Transcription selectively copies only
• certain parts of the genome and makes
• one to several hundred, or even
• thousand, copies of any given section
• of the genome.
• Replication must copy the entire genome and
  do so only once every cell division.

• Similarity
• Both involve enzymes that synthesize a new strand
  of nucleic acid complementary to a
• DNA template strand.

3
Part 1RNA Polymerases and the Transcription
Cycle

4
RNA Polymerases Come in Different Forms,but
Share Many Features

• RNA Polymerase performs essentially the same
  reaction in all cells.
• From bacteria to mammals, the cellular RNA
  Polymerases are made up of multiple subunits.
  (Table 12-1)
• Bacteria have only a single RNA polymerase, while
  in eukaryotic cells there are three RNA Pol I,
  II and III.
• Pol II is the most studied of these enzymes, and
  is responsible for transcribing most
  genes----indeed, essentially all protein-encoding
  genes.
• Pol I transcribes the large ribosomal RNA
  precursor gene.
• Pol III transcribes tRNA genes, some small
  nuclear RNA genes, and the 5S rRNA gene.

5

• The bacterial RNA polymerase core enzyme alone is
  capable of synthesizing RNA and comprises two
  copies of the asubunit and one each of theß, ß
  and?subunits.

b
a
b
a
w
6
Figure 12-2 Comparison of the crystal structures
of prokaryotic and eukaryotic RNA polymerases
The same color indicate the homologous subunits
of the two enzymes
7
Overall ,the shape of each enzyme resembles a
crab claw
pincer
Active center cleft
pincer
8
Transcription by RNA Polymerase Proceeds in a
Series of Steps

• To transcribe a gene, RNA polymerase proceeds
  through a series of well-defined steps which are
  grouped into three phases
• Initiation
• Elongation
• Termination

9
Initiation

• A promoter is the DNA sequence that initially
  binds the RNA polymerase. Once formed, the
  promoter-polymerase complex undergoes structural
  changes required for initiation to proceed.
• DNA at the transcription site unwinds and a
  bubble forms.
• Like replication, transcription occurs in a 5 to
  3 direction.
• Only one of the DNA stands 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.

10
Elongation

• Once the RNA polymerase has synthesized a short
  stretch of RNA (approximately 10 bases),
  transcription shifts into the elongation phase.
• This transition requires further conformational
  change in polymerase that leads it to grip the
  template more firmly.
• Additional tasks of the RNA polymerase
• ?Unwinds the DNA in front and re-anneals it
  behind
• ?Dissociates the growing RNA chain from the
  template
• ?Performs proofreading functions.

11
Termination

• Once the polymerase has transcribed the length of
  the gene (or genes), it must stop and release the
  RNA product. This step is called termination.
• In some cells there are specific,
  well-characterized, sequences that trigger
  termination in others it is less clear what
  instructs the enzyme to cease transcribing and
  dissociate from the template.

12
Figure 12-3 The phases of the transcription
cycle initiation, elongation and termination
elongation
initiation
Termination
13
Transcription Initiation Involves Three Defined
Steps

• The first step is the initial binding of
  polymerase to a promoter to form 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 14bp
  around the start site to form the transcription
  bubble.
• Once an enzyme gets further than the 10bp, it is
  said to have escaped the promoter. At this point
  it has formed a stable ternary complex,
  containing enzyme, DNA, and RNA. This is the
  transition to elongation .

14
Part 2The Transcription Cycle in Bacteria

15
Bacterial Promoters Vary in Strength and
Sequence, but Have Certain Defining Features

• In cells, polymerase initiates transcription only
  at promoters. It is the addition of an initiation
  factor called s that converts core enzyme into
  the form that initiates only at promoters. That
  form of the enzyme is called the RNA polymerase
  holoenzyme.
• holoenzyme sfactor core enzyme

16
Figure 12-4 RNA polymerase holoenzyme T.aquaticus.
17
The predominant sfactor in E.coli is s70

• Promoter recognized by s70 contains two conserved
  sequences (-35 and 10 regions/elements)
  separated by a non-specific stretch of 17-19
  nucleotides.
• The 1 position is designated as the
  transcription start site.

18
Figure 12-5 Features of bacterial promoters (a)

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

19
Figure 12-5 Features of bacterial promoters (b)

• UP-element is an additional DNA elements that
  increases polymerase binding by providing an
  additional specific interaction between the RNA
  polymerase and the DNA.

20
Figure 12-5 Features of bacterial promoters (c)

• Another class of s70 promoter lacks a 35 region
  and has an extended 10 element compensating
  for the absence of 35 region

21
The sFactor Mediates Binding Polymerase to the
Promoter

• The s70 factor can be divided into four regions
  called sregion 1 through sregion 4.

Figure 12-6 Regions of s
22

• Region 2 recognizes -10 element Region 4
  recognizes -35 element
• Region 3 recognizes the extended -10
  element

23
Two helices within 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 contacts with DNA backbone.

24
The interaction with -10 region is less
well-characterized and is more complicated.

• Reasons
• The -10 region is within that element that DNA
  melting is initiated in the transition from the
  closed to open complex.
• The a helix recognizing 10 can interact with
  bases on the nontemplate strand to stabilize the
  melted DNA.

25
The extended -10 element, where present, is
recognized by an a helix in sregion 3.

• The helix makes contact with the two specific
  base pairs that constitute that element.

26
The UP-element is recognized by a carboxyl
terminal domain of the a-subunit (aCTD), but not
by s factor.
Figure 12-7 s and a subunits recruit RNA
polymerase core enzyme to the promoter
27
Transition to the Open Complex Involves
Structural Changes in RNA Polymerase and in the
Promoter DNA

• In the case of the bacterial enzyme bearing s70
  ,this transition, often called isomerization,
  does not require energy, but is the result of a
  spontaneous conformational change in the
  DNA-enzyme complex to a more energetically
  favorable form.

28
There are five channels into the RNA polymerase
holoenzyme.
Figure 12-8 Channels into and out of the open
complex
29

• Within the active center cleft, the DNA strands
  separate from position 3.
• The nontemplate strand goes through the
  nontemplate-strand channel and travels across the
  surface of the enzyme.
• The template strand goes through the
  template-strand channel.
• The double helix re-forms at -11 in the upstream
  DNA behind the enzyme.

30
Two striking structural changes in the enzyme
upon isomerization

• First, the pincers at the front of the enzyme
  clamp down tightly on the downstream DNA.
• Second, there is a major shift in the position of
  the N-terminal region of s. In the closed
  complex, s region 1.1 is in the active center in
  the open complex, the region 1.1 shift to the
  outside of the center, allowing DNA access to the
  cleft.

31
Transcription is Initiated by RNA Polymerase
without the Need for a Primer

• The initiation requires that the initiating
  ribonucleotide (usually an A) be brought into the
  active site and held stably on the template while
  the next NTP is presented with correct geometry.
• Thus the enzyme has to make specific interactions
  with the initiating ribonucleotide, holding it
  rigidly in the correct orientation to allow
  chemical attack on the incoming NTP.

32
RNA Polymerase Synthesizes Several Short RNAs
before Entering the Elongation Phase

• Once ribonucleotides enter the active center
  cleft and RNA synthesis begins, there follows a
  period called abortive initiation.
• Abortive initiation the enzyme synthesizes and
  releases short RNA molecules less than 10
  nucleotides in length.
• Once a polymerase manages to make an RNA longer
  than 10bp, a stable ternary complex is formed.
  This is the start of the elongation phase.

33
Structural barrier for the abortive initiation

• The 3.2 region of s factor lies in the middle of
  the RNA exit channel in the open complex.
• Ejection of this region from the channel (1)
  is necessary for further RNA elongation (2)
  takes the enzyme several attempts

34

35
The Elongating Polymerase Is a Processive Machine
that Synthesizes and Proofreads RNA

• Synthesizing by RNA polymerase
• 1. DNA enters the polymerase between the
  pincers.
• 2. Strand separation in the catalytic cleft.
• 3. NTP addition.
• 4. RNA product spooling out (Only 8-9 nts of the
  growing RNA remain base-paired with the DNA
  template at any given time).
• 5. DNA strand annealing in behind.

36
Proofreading by RNA polymerase

• Pyrophosphorolytic editing the enzyme catalyzes
  the removal of an incorrectly inserted
  ribonucleotide by reincorporation of PPi.
• Hydrolytic editing the enzyme backtracks by one
  or more nucleotides and removes the
  error-containing sequence. This is stimulated by
  Gre factor, a elongation stimulation factor.

37
Transcripyion Is Terminated by Signals within the
RNA Sequence

• Sequences called terminators trigger the
  elongating polymerase to dissociate from the DNA
  and release the RNA chain it has made.
• In bacteria, terminators come in two types
• Rho-independent and Rho-dependent.

38

• Rho-independent terminators, also called
  intrinsic terminators, consist of two sequence
  elements
• a short inverted repeat (of about 20
  nucleotides)
• a stretch of about 8 AT base pairs.

39
Figure 12-9 Sequence of a rho-independent
terminator
40

• When polymerase transcribes an inverted repeat
  sequence, the resulting RNA can form a stem-loop
  structure (often called a hairpin) by
  base-pairing with itself.
• The hairpin is believed to cause termination by
  disrupting the elongation complex.
• The hairpin only works as an efficient
  terminator when it is followed by a stretch of
  AU base pairs.
• AU base pairs are the weakest of all base pairs,
  so they can make the dissociation more easier.

41
Figure 12-10 Transcription termination
42
Rho-dependent terminators

• Have less well-characterized RNA elements, and
  requires Rho protein for termination.
• Rho is a ring-shaped single-stranded RNA binding
  protein, like SSB, and has six identical
  subunits.
• Rho binding can wrest the RNA from the
  polymerase-template complex using the energy from
  ATP hydrolysis.
• Rho binds to rut sites (for Rho Utilization) and
  does not bind the transcripts that are being
  translated.

43
Figure 12-11 The ?transcription termination
factor
44
Part 3 Transcription in Eukaryotes

45
A little comparison
Items Eukaryotes prokaryotes
RNA Polymerase Pol I, II and III Core enzyme
Additional initiation factor General transcription factors (GTFs) sfactors
46
In addition to the RNAP and GTFs, in vivo
transcription also requires

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

47
RNA Polymerase II Core Promoters Are Made up of
Combinations of four Different Sequence Elements

• The eukaryotic core promoter the minimal set of
  sequence elements required for accurate
  transcription initiation by the Pol II machinery
  in vitro.
• A core promoter is typically about 40 nucleotides
  long, extending either upstream or downstream of
  the transcription start site.
• Four elements in Pol II core promoters
• The TFIIB recognition element (BRE)
• The TATA element (or box)
• The initiator (Inr)
• The downstream promoter element (DPE)

48
Figure 12-12 Pol II core promoter
The figure shows the position of various DNA
elements relative to the transcription start
site. Below are the consensus sequence for each
element and above are the names of the general
transcription factors that recognize them.
49
Regulatory sequences are also required for
efficient transcription in vivo besides the core
promoter.

• These elements include
• Promoter proximal elements
• Upstream activator sequences (UASs)
• Enhancers
• A series of repressing elements called
• silencers, boundary elements, and
  insulators.
• All these DNA elements bind regulatory
  proteins, which help or hinder transcription from
  the core promoter.

50
RNA Polymerase II Forms a Pre-Initiation Complex
with General Transcription Factors at the Promoter

• Pre-initiation complexThe complete set of
  general transcription factors and polymerase
  bound together at the promoter and poised for
  initiation.
• The TATA element where pre-initiation complex
  formation begins is recognized by the the general
  transcription factor called TFIID.
• TFIID TBP TAFs

TBP associated factors
TATA binding protein
51

• Upon binding DNA, TBP extensively distorts the
  TATA sequence. The resulting TBP-DNA complex
  provides a platform to recruit other general
  transcription factors and polymerase itself to
  the promoter.
• In vitro, these proteins assemble in the
  following order
• TFIIA, TFIIB, TFIIF together with polymerase,
  TFIIE and TFIIH.

52
Figure 12-13 Transcription initiation by RNA
polymerase II

1. TBP in TFIID binds to the TATA box
2. TFIIA and TFIIB are recruited with TFIIB binding
   to the BRE
3. RNA Pol II-TFIIF complex is then recruited
4. TFIIE and TFIIH then bind upstream of Pol II to
   form the pre-initiation complex
5. Promoter melting using energy from ATP hydrolysis
   by TFIIH )
6. Promoter escapes after the phosphorylation of the
   CTD tail

53
Promoter escape

• In eukaryotes, promoter escape involves the
  phosphorylation of the polymerase.
• The large 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
  .
• Each repeat contains sites for phosphorylation
  by specific kinases including one that is a
  subunit of TFIIH.
• Phosphorylation helps polymerase shed most of the
  general transcription factors and leave them
  behind as it escape the promoter.

54
TBP Binds to and Distorts DNA Using a b Sheet
Inserted into the Minor Groove

• TBP uses an extensive region of b sheet to
  recognize the minor groove of the TATA element.
• This is unusual and the reason for TPBs
  unorthodox recognition mechanism is linked to the
  need for that protein to distort the local DNA
  structure.

55
Figure 12-14 TBP-DNA complex
56
Much of the specificity is imposed by two pairs
of phenylalanine side chains

• The phenylalanine side chains intercalate between
  the base pairs at either end of the recognition
  sequence and drive the strong bend in the DNA.
• AT base pairs are favored because they are more
  readily distorted to allow the initial opening of
  the minor groove.
• There are also extensive interactions between the
  phosphate backbone and basic residues in the b
  sheet, adding to the overall binding energy of
  the interaction.

57
The Other General Transcription Factors also Have
Specific Roles in Initiation

• TAFs.
• Two of them bind DNA elements at the promoter
  (e.g. Inr and DPE).
• Several TAFs have structural homology to histone
  proteins and might bind DNA in a similar manner.
• Another TAF appears to regulate the binding of
  TBP to DNA.

58
TFIIB.

1. A single polypeptide chain, enters the
   pre-initiation complex after TPB.
2. The asymmetric binding of TFIIB to the TBP-TATA
   complex accounts for the asymmetry in the rest
   of the assembly of the pre-initiation complex and
   the unidirectional transcription that results.
3. Bridges the TATA-bound TBP and polymerase.
4. The N-terminal domain inserts into the RNA exit
   channel of Pol II in a manner analogous to s3.2
   in the bacterial case.

59
Figure 12-15 TFIIB-TBP-promoter complex.
60
TFIIF.

1. A two-subunit factor associates with Pol II and
   is recruited to the promoter together with that
   enzyme (and other factors).
2. Binding of Pol II-TFIIF stabilizes the
   DNA-TBP-TFIIB complex.
3. Required before TFIIE and TFIIH are recruited to
   the pre-initiation complex.

61
TFIIE and TFIIH.

• TFIIE Recruits and regulates TFIIH.
• TFIIH 1.Controls the ATP-dependent transition of
  the pre-initiation
  complex to the open complex.
• 2.The largest and most complex
  GTF---it has 9 subunits. Two function as
  ATPases and one is a protain kinase with roles in
  promoter melting and escape.
• 3.Together with other factors,
  the ATPase subunits are also involved in
  nucleotide mismatch repair.

62
In Vivo, Transcription Initiation Requires
Additional Proteins, Including the Mediator
Complex

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

63
Figure 12-16 Assembly of the pre-initiation
complex in presence of Mediator, nucleosome
modifiers and remodelers, and transcriptional
activators.
64
Mediator Consists of Many Subunits, Some
Conserved from Yeast to Human
Figure 12-17 Comparison of the yeast and human
Mediators.
65

• Both include more than 20 subunits, of which 7
  show significant sequence homology.
• Only subunit Srb4 is essential for transcription
  of essentially all Pol II genes in vivo.
• Both are organized in modules.
• RNA Pol II holoenzyme is a putative preformed
  complex
• Pol II mediator some of GTFs

66
A new Set of Factors Stimulate PolI Elongation
and RNA Proofreading

• The transition from the initiation to elongation
  involves the Pol II enzyme shedding most of its
  initiation factors (e.g. GTFs and Mediators) and
  recruiting other factors
• 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.
• 
  

67
Figure 12-18 RNA processing enzymes are recruited
by the tail of polymerase
68
Factors that stimulate elongation

• P-TEFb
• phosphorylates CTD
• Activates hSPT5
• Activates TAT-SF1
• TFIIS
• Stimulates the overall rate of elongation by
  limiting the length of time polymerase pauses.
• Contributes to proofreading by polymerase.

69
Elongating Polymerase Is Associated with a New
Set of Protein Factors Required for Various Types
of RNA Processing

• Once transcribed, eukaryotic RNA has to be
  processed in various ways before exported from
  the nucleus where it can be translated.
• These processing events include

• Capping of the 5 end of the RNA
• Splicing
• Polyadenylation of the 3 end of the RNA.

70

• Elongation, termination of transcription,
  and RNA processing are interconnected to ensure
  their proper coordination.

71
The first RNA processing event is capping.

1. A phosphate group is removed from the 5 of the
   transcript.
2. The GTP is added.
3. The nucleotide is modified by the addition of a
   methyl group.

• The RNA is capped when it is still only
  some 20-40 nucleotides long.

72
Figure 12-19 The structure and formation of the
5 RNA cap
The capping involves the addition of a methylated
guanine joined to the RNA transcript by an
unusual 5-5 linkage involving 3 phosphates.
73
The second event splicing

• After capping, dephosphorylation of Ser5
  within the tail repeats leads to dissociation of
  the capping machinery, and further
  phosphorylation (this time of Ser2 within the
  tail repeats) cause recruitment of the machinery
  needed for RNA splicing.

74
The final event polyadenylation of the 3 end of
the mRNA

• Linked with the termination of transcription.
• The CTD tail is involved in recruiting the
  polyadenylation enzymes.
• The transcribed poly-A signal triggers the
  reactions
• Cleavage of the message.
• Addition of many adenine residues to its 3 end
• Termination of transcription by polymerase.

75
Figure 12-20 Polyadenylation and termination
76
Termination

• The enzyme does not terminate immediately when
  the RNA is cleaved and polyadenylated.
• Rather, it continues to move along the template,
  generating a second RNA molecule that can become
  as long as several hundred nucleotides before
  terminating.
• The polymerase then dissociates from the
  template, releasing the new RNA, which is
  degraded without ever leaving the nucleus.

77
RNA Pol I III Recognize Distinct Promoters ,
Using Distinct Sets of Transcription Factors, but
Still Require TBP

• Pol I transcribes rRNA precursor encoding gene
  (multi-copy gene)
• Pol III transcribes tRNA genes, some small
  nuclear RNA genes and the 5S rRNA genes

78
Pol I promoter recognition Figure 12-21
Pol I promoter region
UBF binds to the upstream half of UCE, bringing
SL1 and stimulating transcription from the core
promoter by recruiting Pol I .
79
Pol III promoter recognition
Figure 12-22 Pol III core promoter

• Pol III promoters come in various forms.
• Most locates downstream of the transcription
  start site.
• Some Pol III promoters consist of Box A Box B,
  some contain Box A Box C, and still others
  contain a TATA element.

TFIIIC binds to the promoter, recruiting
TFIIIB, which in turn recruits Pol III.
80
Figure12-1 Transcription of DNA into RNA
81
Table 12-1 The Subunits of RNA Polymerases