Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
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Part III Expression of the Genome

• Ch 12 Mechanisms of transcription
• Ch 13 RNA splicing
• Ch 14 Translation
• Ch 15 The genetic code

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Chapter 12 Mechanisms of Transcription

• Molecular Biology Course

• RNA polymerase and transcription cycle
• The transcription cycle in bacteria
• Transcription in eukaryotes

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The Central Dogma
Transcription Translation
DNA RNA PROTEIN
replication
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Transcription is very similar to DNA replication
but there are some important differences

• RNA is made of ribonucleotides
• RNA polymerase catalyzes the reaction
• The synthesized RNA does not remain base-paired
  to the template DNA strand
• Less accurate (error rate 10-4)

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• 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?)

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Transcription bubble
Fig 12-1 Transcription of DNA into RNA
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Topic 1 RNA Polymerase and The Transcription
Cycle
CHAPTER12 Mechanisms of Transcription
See the interactive animation
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RNA polymerases come in different forms, but
share many features
RNA polymerase and the transcription cycle

• RNA polymerases performs essentially the same
  reaction in all cells
• Bacteria have only a single RNA polymerases while
  in eukaryotic cells there are three RNA Pol I,
  II and III

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• RNA Pol II is the focus of eukaryotic
  transcription, because it is the most studied
  polymerase, and is also responsible for
  transcribing most genes-indeed, essentially all
  protein-encoding genes
• RNA Pol I transcribe the large ribosomal RNA
  precursor gene
• RNA Pol II transcribe tRNA gene, some small
  nuclear RNA genes and the 5S rRNA genes

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Table 12-1 The subunits of RNA polymerases
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The bacterial RNA polymerase
The core enzyme alone synthesizes RNA
b
a
b
a
w
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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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Crab claw shape of RNAP (The shape of DNA pol
is__)
Active center cleft
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There are various channels allowing DNA, RNA and
ribonucleotides (rNTPs) into and out of the
enzymes active center cleft
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Transcription by RNA polymerase proceeds in a
series of steps
RNA polymerase and the transcription cycle

• Initiation
• Elongation
• Termination

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Initiation

• Promoter the DNA sequence that initially binds
  the RNA polymerase
• The structure of promoter-polymerase complex
  undergoes structural changes to proceed
  transcription
• DNA at the transcription site unwinds and a
  bubble forms
• Direction of RNA synthesis occurs in a 5-3
  direction (3-end growing)

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

• Once the RNA polymerase has synthesized a short
  stretch of RNA ( 10 nt), transcription shifts
  into the elongation phase.
• This transition requires further conformational
  change in polymerase that leads it to grip the
  template more firmly.
• Functions synthesis RNA, unwinds the DNA in
  front, re-anneals it behind, dissociates the
  growing RNA chain

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Termination

• After the polymerase transcribes the length of
  the gene (or genes), it will stop and release the
  RNA transcript.
• In some cells, termination occurs at the specific
  and well-defined DNA sequences called
  terminators. Some cells lack such termination
  sequences.

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Fig 12-3-Elongation and termination
Elongation
Termination
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Transcription initiation involves 3 defined steps
RNA polymerase and the transcription cycle

• Forming closed complex
• Forming open complex
• Promoter escape

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Closed complex

• The initial binding of polymerase to a promoter
• DNA remains double stranded
• The enzyme is bound to one face of the helix

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Open complex

• the DNA strand separate over a distance of 14 bp
  (-11 to 3 ) around the start site (1 site)
• Replication bubble forms

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Stable ternary complex
RNA polymerase and the transcription cycle

• The enzyme escapes from the promoter
• The transition to the elongation phase
• Stable ternary complex
• DNA RNA enzyme

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Topic 2The transcription cycle in bacteria
CHAPTER12 Mechanisms of Transcription
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2-1 Bacterial promoters vary in strength and
sequences, but have certain defining features
The transcription cycle in bacteria
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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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Promoters recognized by E. coli s factor

• The predominant s factor 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 nt.
• Position 1 is the transcription start site.

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Fig 12-5a bacterial promoter
The distance is conserved

• 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

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BOX 12-1 Figure 1
Consensus sequence of the -35 and -10 region
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• Promoters with sequences closer to the consensus
  are generally stronger than those match less
  well. (What does stronger mean?)
• The strength of the promoter describes how many
  transcripts it initiates in a given time.

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Fig 12-5b bacterial promoter
Confers additional specificity
UP-element is an additional DNA elements that
increases polymerase binding by providing the
additional interaction site for RNA polymerase
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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 s factor mediates binding of polymerase
to the promoter
The transcription cycle in bacteria

• The s70 factor comprises four regions called s
  region 1 to s region 4.

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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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Binding of 35 Two helices within region 4 form a
common DNA-binding motif, called a
helix-turn-helix motif
One helix inserts into the DNA major groove
interacting with the bases at the 35 region. The
other helix lies across the top of the groove,
contacting the DNA backbone
Fig 5-20 Helix-turn-helix DNA-binding motif
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Interaction with 10 is more elaborate (??) and
less understood

• The -10 region is within DNA melting region
• The a helix recognizing 10 can interacts with
  bases on the non-template strand to stabilize the
  melted DNA.

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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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2-3 Transition to the open complex involves
structural changes in RNA polymerase and in the
promoter DNA
The transcription cycle in bacteria

• This transition is called Isomerization (???)

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• For s70 containing RNA polymerase,
  isomerization is a spontaneous conformational
  change in the DNA-enzyme complex to a more
  energetically favorable form. (No extra energy
  requirement)

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Change of the promoter DNA

• the opening of the DNA double helix, called
  melting, at positions -11 and 3.

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The striking structural change in the polymerase

• 1. the b and b pincers down tightly on the
  downstream DNA
• 2. A major shift occurs in the N-terminal region
  of s (region 1.1) shifts. 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

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Fig 12-8 channels into and out of the open complex
NTP uptake channel is in the back
DNA entering channel
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Transcription is initiated by RNA polymerase
without the need for a primer
The transcription cycle in bacteria

• Initiation requires
• The initiating NTP (usually an A) is placed in
  the active site
• The initiating ATP is held tightly in the correct
  orientation by extensive interactions with the
  holoenzyme

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RNA polymerase synthesizes several short RNAs
before entering the elongation phase
The transcription cycle in bacteria

• Abortive initiation the enzyme synthesizes and
  releases short RNA molecules less than 10 nt.

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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

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Fig 12-8 channels into and out of the open complex
NTP uptake channel is in the back
DNA entering channel
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The elongating polymerase is a processive machine
that synthesizes and proofreads RNA
The transcription cycle in bacteria
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Synthesizing by RNA polymerase

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

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Proofreading by RNA polymerase

• Pyrohosphorolytic (?????)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.

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Transcription is terminated by signals within the
RNA sequence
The transcription cycle in bacteria

• Terminators the sequences that trigger the
  elongation polymerase to dissociate from the DNA
• Rho-dependent (requires Rho protein)
• Rho-independent, also called intrinsic (??)
  terminator

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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 12-10 transcription termination
Weakest base pairing AU make the dissociation
easier
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Rho (r) -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
• Rho binding can wrest (??) the RNA from the
  polymerase-template complex using the energy from
  ATP hydrolysis
• Rho binds to rut (r utilization) RNA sites
• Rho does not bind the translating RNA

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Fig 12-11 the r transcription terminator
RNA tread trough the ring
Hexamer, Open ring
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Topic 3transcription in eukaryotes
CHAPTER12 Mechanisms of Transcription
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Comparison of eukaryotic and prokaryotic RNA
polymerases

• Eukaryotes Three polymerase transcribes
  different class of genes Pol I-large rRNA genes
  Pol II-mRNA genes Pol III- tRNA, 5S rRNA and
  small nuclear RNA genes (U6)
• Prokaryotes one polymerase transcribes all genes

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Comparison of eukaryotic and prokaryotic promoter
recognition

• Eukaryotes general transcription factors (GTFs).
  TFI factors for RNAP I, TFII factors for RNAP II
  and TFIII factors for RNAP III
• Prokaryotes s factors

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• In addition to the RNAP and GTFs, in vivo
  transcription also requires
• Mediator complex
• DNA-binding regulatory proteins
• chromatin-modifying enzymes
• Why??

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RNA polymerase II core promoters are made up of
combinations of 4 different sequence elements
The transcription in eukaryotes

• Eukaryotic core promoter (40 nt) the minimal
  set of sequence elements required for accurate
  transcription initiation by the Pol II machinery
  in vitro

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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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Regulatory sequences

• The sequence elements other than the core
  promoter that are required to regulate the
  transcription efficiency
• Those increasing transcription
• Promoter proximal elements
• Upstream activator sequences (UASs)
• Enhancers
• Those repressing elements silencers, boundary
  elements, insulators (???)

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RNA Pol II forms a pre-initiation complex with
GTFs at the promoter
The transcription in eukaryotes

• The involved GTFIIs (general transcription factor
  for Pol II)
• TFIIDTBP (TATA box binding protein) TAFs (TBP
  association factors)
• TFIIA, B, F, E, H

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

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Promoter escape

• Stimulated by phosphorylation of the CTD
  (C-terminal domain) tail of the RNAP II
• CTD contains the heptapeptide repeat
  Tyr-Ser-Pro-Thr-Ser-Pro-Ser
• Phosphorylation of the CTD tail is conducted
  by a number of specific kinases including a
  subunit of TFIIH

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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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• AT base pairs (TATA box) are favored because
  they are more readily distorted to allow initial
  opening of the minor groove

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The other GTFs also have specific roles in
initiation
The transcription in eukaryotes

• 10 TAFs (1) two of them bind DNA elements at
  the promoter (Inr and DPE) (2) several are
  histone-like TAFs and might bind to DNA similar
  to that histone does (3) one regulates the
  binding of TBP to DNA

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• TFIIB (1) a single polypeptide chain, (2)
  asymmetric binding to TBP and the promoter DNA
  (BRE), (3)bridging TBP and the polymerase, (4)
  the N-terminal inserting in the RNA exit channel
  resembles the s3.2 .

Fig 12-13 TFIIB-TBP-promoter complex
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• TFIIF (1) a two subunit factor, (2) binding of
  Pol II-TFIIF stabilizes the DNA-TBP-TFIIB
  complex, which is required for the followed
  factor binding
• TFIIE recruits and regulates TFIIH
• TFIIH (1) controls the ATP-dependent transition
  of the pre-initiation complex to the open
  complex, (2) contains 9 subunits and is the
  largest GTF two functions as ATPase and one is
  protein kinase. (3) important for promoter
  melting and escape. (4) ATPase functions in
  nucleotide mismatch repair, called
  transcription-coupled repair.

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in vivo, transcription initiation requires
additional proteins
The transcription in eukaryotes

• The mediator complex
• Transcriptional regulatory proteins
• Nucleosome-modifying enzymes
• To counter the real situation that the DNA
  template in vivo is packed into nucleosome and
  chromatin

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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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Mediator consists of many subunits, some
conserved from yeast to human
The transcription in eukaryotes

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

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Fig 12-17 comparison of the yeast and human
mediators
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Eukaryotic RNA Pol II holoenzyme is a putative
preformed complex Pol II mediator some of
GTFs
Prokaryotic RNA Polymerase holoenzyme core
polymerase s factor
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A new set of factors stimulate Pol II elongation
and RNA proofreading
The transcription in eukaryotes
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Transition from the initiation to elongation
involves the Pol II enzyme shedding (??) most of
its initiation factors (GTF and mediators) and
recruiting other factors (1) Elongation
factors factors that stimulate elongation, such
as TFIIS and hSPT5. (2) RNA processing (RNA ??)
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 12-18 RNA processing enzymes are recruited by
the tail of polymerase
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Some elongation factors

• P-TEFb
• phosphorylates CTD
• Activates hSPT5
• Activates TAT-SF1
• TFIIS
• Stimulates the overall rate of elongation by
  resolving the polymerase pausing
• Proofreading

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Elongation polymerase is associated with a new
set of protein factors required for various types
of RNA processing
The transcription in eukaryotes

• RNA processing
• Capping of the 5 end of the RNA
• Splicing of the introns (most complicated)
• Poly adenylation (?????) of the 3 end

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Elongation, termination of transcription, and RNA
processing are interconnected/ coupled (???) to
ensure the coordination (???) of these events

• Evidence this is an overlap in proteins
  involving in those events
• 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 poly(A) tail

• Increased mRNA stability
• Increased translational efficiency
• Splicing of last intron

AAAAAA
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Function of 5cap

• Protection from degradation
• Increased translational efficiency
• Transport to cytoplasm
• Splicing of first intron

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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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3 end polyadenylation

• 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 poly-A
• Termination of transcription

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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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• What terminates transcription by polymerase?

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Models to explain the link between
polyadenylation and termination (see the
animation on your CD)

• Model 1 The transfer of the 3 processing enzyme
  to RNAP II induces conformational changeRNAP II
  processivity reducesspontaneous termination
• Model 2 absence of a 5cap on the second RNA
  moleculerecognized by the RNAP II as
  improperterminate transcription

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RNA Pol I III recognize distinct promoters ,
using distinct sets of transcription factors, but
still require TBP
The transcription in eukaryotes

• Pol I transcribes rRNA precursor encoding gene
  (multi-copy gene)
• Pol III transcribes tRNA genes, snRNA genes and
  5S rRNA genes

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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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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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Key points of the chapter

• RNA polymerases (RNAP, ????????) and
  transcription cycle (Initiation is more
  complicate, details in bacteria)
• Transcription cycle in bacteria
• (1) promoters (elements), s factor (4 domains),
  aCTD, abortive initiation (why?)
• (2) Structures accounting for formation of the
  closed complex, transitions to open complex and
  then stable ternary complex.
• (3) Elongation and editing by polymerase (10-4)
• (4) Termination Rho-independent and
  Rho-dependent mechanism

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• Transcription cycle in eukaryotes
• Promoters (elements), general transcription
  factors (GTF),
• RNAP II transcription
• ---the roles of GTFs and the CTD tail of RNAP II
  in promoter recognition, formation of the
  pre-initiation complex, promoter melting,
  promoter escape
• ---in vivo requires mediator complex, nucleosome
  modifying enzymes and transcription regulatory
  proteins.
• ---elongation and proofreading involve a new set
  of GTFs (What)
• ---coupled with RNA processing (How)

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• RNAP I and III transcription
• ---GTFs and promoter recognition, formation of
  the initiation complex

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Homework
See the animations Complete all the excises on
your study CD. Enjoy it