1. Gene expression

1.3. Eukaryotic Transcription

Initiation of Transcription by RNA Polymerase II
Initiation-Formation of the Transcription complex
  • The first step in formation of a transcription complex is the binding of a general transcription factors called TFIID to the TATA box.TFIID is itself composed of multiple subunits,  including the TATA binding protein . This binds specifically to the TATAA consensus sequence .and 10-12 other polypeptides, called TBP-associated factors (TAFs).
  • TBP then binds a second general transcription factor (TFIIB) forming a TBP-TFIIB complex at the along with TF II F at the promoter
  • Then RNA polymerase II binds
  • This is followed by the binding of TFIIE and TFIIH.
  • TFIIH is a multi subunit factor. It plays  two important roles. First, two subunits of TFIIH are helicases, which unwind DNA  around the initiation site. These subunits of TFIIH are also required for Nucleotide excision repair .Another subunit of TFIIH is a  Protein kinase . This phosphorylates repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. 
  • Once the complex is assembled, RNA polymerase can bind to its upstream sequence.
  • When bound along with the transcription factors, RNA polymerase is phosphorylated. This releases part of the protein from the DNA .  The transcription initiation complex  gets activated and places RNA polymerase in the correct orientation to begin transcription.
  • Then the double-stranded DNA in the transcription start region  is unwound . The RNA Polymerase II is then  positioned at the +1 initiation nucleotide and starts new RNA strand synthesis.

Elongation

  • RNA Polymerase  clears or “escapes” the promoter region and leaves most of the transcription initiation proteins behind.

  • RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction .The synthesis of new RNA strands takes place in the 5′ to 3′ direction. i.e.,  new nucleotides are added to the 3′ end of the growing RNA strand.

  • RNA Polymerases unwind the double stranded DNA ahead of them and rewinds the unwound DNA behind them.
  • RNA strand synthesis occurs in a transcription bubble of about 25 unwound DNA base pairs.
  • About 8 nucleotides of newly-synthesized RNA remain base paired to the template DNA. The rest of the RNA molecules falls off the template  and this  allows the DNA behind it to rewind.
Termination
  • The termination of transcription is different for the three different eukaryotic RNA polymerases.
  • RNA Polymerase I contain a specific sequence of base pairs -11 bp long in humans  :  18 bp in mice .  This sequence is recognized by a termination protein called TTF-1 (Transcription Termination Factor for RNA Polymerase I.).  This protein binds the DNA at its recognition sequence and blocks further transcription, causing the RNA Polymerase I to detach from the template DNA strand and  releases the newly-synthesized RNA.
  • RNA Polymerse II lack any specific signals or sequences that direct RNA Polymerase II to terminate at specific locations. The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. The cleavage site occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA.
  •  The upstream portion of the transcript is released .
  • The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it  disengage the polymerase from its DNA template strand, finally terminating transcription.
  • A protein called CPSF in humans binds the AAUAAA sequence and a protein called CstF in humans binds the GU-rich sequence. CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides downstream from the AAUAAA site. The Poly(A) Polymerase enzyme  catalyze the addition of a 3′ poly-A tail on the pre-mRNA .
  • The RNAs transcribed by RNA Polymerase III have a short stretch of four to seven U’s at their 3′ end. This somehow triggers RNA Polymerase III to both release the nascent RNA and disengage from the template DNA strand.