🖨️ Lecture 4: Eukaryotic Transcription Initiation

Transcription Initiation at Eukaryotic Promoters

Some Things to Keep in Mind
 At most TATA and or Initiator Inr containing promoters
  Transcription initiates and proceeds in one direction

 At CpG island promoters
  Transcription initiates in both directions
  Polymerase stalls and falls off in one direction
  Transcription proceeds in the other direction only

Textbook Note
 Current textbook has no good CpG island initiation figure
 Figure shown is from the 8th edition

RNA Polymerase II
 Loaded onto promoters by GTFs
 Pauses downstream of the initiation site

CpG Island Near the Transcriptional Initiation Site of the Human APRT Gene

APRT Gene
 APRT stands for Adenine Phosphoribosyl Transferase

CpG Island Location
 CpG islands are located near the transcriptional initiation site
 CG repeats are highly concentrated near the start codon

Relationship to Transcription
 The start codon is located close to the transcription start site
 High CG density is associated with transcription initiation

CpG Islands and Gene Transcription

CpG Island Promoters
 CpG islands occupy the promoters of about 70 percent of vertebrate genes
 These genes are often essential
 They are transcribed at a low but constant rate

Chromatin Features
 CpG rich DNA contains fewer nucleosomes
 Fewer nucleosomes make DNA easier to transcribe

Transcription Initiation
 At CpG islands transcription initiates at any position within the island
 Initiation site is not precisely defined
 Transcription initiates in both directions
 Transcription proceeds only toward the Open Reading Frame ORF
 ORF is the DNA region that codes for protein

Textbook Note
 Deamination and stability of methylated C in CpG islands in mammals is not important for this class

Bi Directional Transcription at CpG Islands

Experimental Approach
 RNA is labeled with Br UTP
 Br UTP is a UTP analogue
 Labeling is done after ChIP using an RNA Pol II antibody
 RNA is then sequenced

Results
 Plot shows intensity of transcription across the analyzed locus
 RNA synthesis initiates in both directions

Key Term
 TSS means Transcription Start Site

Meaning of the Results

Transcript Peaks
 Transcripts peak at approximately +50 and −250

Interpretation
 RNA Pol II initiates transcription in both directions
 RNA Pol II then pauses before elongating further in the sense direction

Key Term
 TSS means Transcription Start Site

Meaning of the Experiment

CpG Island Transcription
 At CpG islands equal numbers of RNA polymerase initiate in both sense and antisense directions

Sense Direction
 Sense transcripts pause before elongating further

Antisense Direction
 Antisense transcripts pause at the opposite end of the CpG island
 They do not continue transcription

Figure 8-12 Promoter Type Analysis

Genes Shown
 Hsd17b12: Hydroxysteroid 17β Dehydrogenase 12
 Rpl6: Ribosomal Protein L6

Question
 What type of promoter does Hsd17b12 have
 What type of promoter does Rpl6 have

Experimental Overview
 Cells treated with formaldehyde to cross-link DNA and proteins
 Chromatin sheared by sonication
 Antibody to RNA Pol II added
 Paused polymerase and nascent RNA captured
 Pol II–DNA complexes immunoprecipitated
 Cross-links reversed
 DNA sequenced

What the Figure Shows
 RNA Pol II signal across each gene
 Direction of transcription initiation is visible

Interpretation
 Bidirectional initiation → CpG island promoter
 Unidirectional initiation → TATA or Inr promoter

Assembly of Pre-Initiation Complex on TATA-Containing Promoters

Promoter Elements
 Promoter elements such as the TATA box or Initiator direct General Transcription Factors to bind DNA

Unbound Promoter
 Promoter DNA contains a TATA box
 TFIID binds first
  TFIID contains TBP and TAFs
  TBP binds the TATA box

Upstream Promoter Complex
 TFIIA and TFIIB bind to stabilize TBP on DNA
 This forms the upstream promoter complex

Core Pre-Initiation Complex Core PIC
 RNA Polymerase II binds with TFIIF
  Pol II contains a CTD tail
 TFIIE and TFIIH bind
 Upstream and downstream DNA are positioned
 This forms the closed PIC

Open Pre-Initiation Complex Open PIC
 TFIIH uses ATP
 DNA is unwound forming a transcription bubble

Initially Transcribing Complex
 NTPs are added
 Nascent RNA is synthesized
 Initiation factors are present

Elongation Complex
 RNA Polymerase II escapes the promoter
 Elongation factors associate
 5′ cap is added to the RNA

Unbound Promoter and Closed Pre-Initiation Complex

Unbound Promoter
 Promoter DNA contains a TATA box
  Consensus shown as ΥΥΥΛΛΑ TATA ΥΥΥ
 TFIID binds first
  TFIID is a multi protein complex
  TBP means TATA Binding Protein
  TAFs are TBP Associated Factors

Early Factor Binding
 TFIIB binds after TFIID
 TFIIA binds to stabilize the complex
 This forms the upstream promoter complex

RNA Polymerase II Recruitment
 RNA Polymerase II is recruited with TFIIF
 Pol II contains a CTD
  CTD means Carboxy Terminal Domain
 CTD is non phosphorylated at this stage
 Non phosphorylated CTD contacts several GTFs

Core Pre-Initiation Complex Core PIC
 TFIIE and TFIIH are recruited
 TFIIH functions as a kinase and a DNA helicase
 Upstream and downstream DNA are positioned
 This state is called the Closed PIC

Key Term
 PIC means Pre-Initiation Complex

Closed PIC to Elongation

Closed PIC
 TFIIH uses ATP
 TFIIH helicase activity opens the DNA double helix

Open PIC
 DNA opening creates a transcription bubble

Initially Transcribing Complex
 NTPs are added
 Nascent RNA is synthesized
 RNA Polymerase II initiates transcription
 Initiation factors are present

CTD Phosphorylation
 TFIIH kinase activity phosphorylates the CTD
 Phosphorylation releases RNA Polymerase II from the promoter

Elongation Complex
 Elongation factors associate
 RNA synthesis continues
 5′ cap is added to the RNA

Transition from Initiation to Elongation (Figure 8-3)

Initiation State
 RNA Polymerase II assembles with General Transcription Factors
 Mediator and activators are present
 Pol II begins transcription
 Nascent transcript is produced

Pausing
 RNA Polymerase II pauses shortly after initiation
 Pausing is stabilized by NELF and DSIF
 Polymerase remains near the promoter

Release from Pausing
 P-TEFb is recruited
 P-TEFb phosphorylates factors associated with Pol II
 NELF dissociates
 DSIF becomes a positive elongation factor

Elongation
 RNA Polymerase II transitions into productive elongation
 Elongation factors associate
 Additional Pol II molecules can initiate
 Gene is transcriptionally on

Scaffold
 Some General Transcription Factors remain at the promoter
 This scaffold allows repeated rounds of transcription

Elongation Factors and Polymerase Pausing

Last Step of Initiation (Fig 8-13)
 TFIIH kinase phosphorylates the CTD
 Phosphorylation releases RNA Polymerase II from the promoter

CTD Phosphorylation
 Initial phosphorylation of the CTD occurs by TFIIH kinase
 This marks the transition away from the promoter

Early Elongation and Pausing
 After CTD phosphorylation RNA Polymerase II moves downstream of the initiation site
 Two negative elongation factors bind
 NELF associates with RNA Polymerase II
 DSIF associates with RNA Polymerase II
 These factors pause the polymerase

Elongation Complex
 Elongation factors are present
 Initiation factors dissociate
 5′ cap is added to the nascent RNA

Key Terms
 NELF means Negative Elongation Factor
 DSIF means DRB Sensitivity Inducing Factor

Release into Productive Elongation

P-TEFb Kinase
 Another kinase P-TEFb also called CDK9 Cyclin T
 P-TEFb means Positive Transcription Elongation Factor b

Further Phosphorylation
 P-TEFb further phosphorylates the CTD of RNA Polymerase II
 P-TEFb also phosphorylates NELF
 RNA Polymerase II with fully phosphorylated CTD is released

Elongation Factor Switch
 NELF dissociates from the polymerase
 DSIF switches from negative to positive elongation factor

Positive Elongation Factors
 PAF joins the polymerase
 Spt16 joins the polymerase

Gene Activation
 RNA Polymerase II moves into productive elongation
 Nascent transcript continues to grow
 Gene is transcriptionally on

Promoter Scaffold
 Some General Transcription Factors remain at the promoter
 This scaffold allows additional Pol II to initiate

Switch from Initiation to Pausing to Elongation (Fig 8-14)

Paused Elongation Complex
 RNA Polymerase II is paused
 DSIF is associated
 NELF is associated
 CTD is not fully phosphorylated

Activated Elongation Complex
 P-TEFb acts on the complex
 CTD is further phosphorylated
 NELF dissociates
 DSIF remains and functions positively

Additional Elongation Factors
 PAF associates with RNA Polymerase II
 SPT6 associates with RNA Polymerase II
 SPT6 removes nucleosomes from upstream DNA

Key Terms
 PAF means Polymerase Associated Factor
 SPT6 means Suppressor of Ty 6

HIV Transcriptional Elongation and Viral Latency (Fig 8-15)

Early Transcription
 The 5′ end of a short HIV RNA is synthesized
 RNA Polymerase II pauses shortly after initiation

TAR Structure
 The 5′ end contains a secondary RNA structure called TAR
 TAR holds the paused polymerase
 TAR inhibits Cdk9 Cyclin T also called P-TEFb

Tat Protein
 HIV encodes a protein called Tat
 Tat binds to TAR
 Under cell stress paused polymerase is released
 This allows some Tat protein to be produced

Release of Pausing
 Tat bound to TAR activates CDK9 Cyclin T kinase
 Activated P-TEFb releases paused RNA Polymerase II

Viral Latency
 HIV remains dormant when polymerase is paused
 Stress activates transcription
 Activated virus kills T cells and abolishes immunity