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Eukaryotic RNAp and Promoters
Eukaryotic RNAp and Promoters
■ Three polypeptides, Rpb1 (named from RNA Pol B), Rpb2, Ppb3 are
absolutely required for enzyme activity
■ These are homologous to β’- β-, and α-subunits
■ Both PRpb1 and β’ –subunit binds DNA
■ Rpb2 and β-subunit are at or near the nucleotide-joining active site
■ Rpb3 does not resemble α-subunit
* There is one 20-amino acid subunit of great similarity
* 2 subunits are about same size, same stoichiometry
* 2 monomers per holoenzyme
Common Subunits
■ During the translocation step, the RNA-DNA hybrid moves one base
pair to left, bringing new template strand nucleotide into the active
site. Simultaneously, the bridge helix bends (green dot), remaining
close to the end of the RNA.
■ When the bridge helix returns to the straight state (arrow at left), it
reopens the active site so another nucleotide can enter.
3-D Structure – RNA Polymerase II in
the Post translocation State
■ X-ray crystallography has shown the lid of Rpb 1 interacts with the
DNA-RNA hybrid to force the hybrid open after base pair8
■ The lid then interacts with bases of the nascent RNA to keep the
hybrid melted beyond base pair-8
■ The rudder of Rpb1 collaborates with lid to keep the hybrid melted by
interacting with bases -9 and -10
■ Fork loop 1 of Rpb2 interacts with bases -5, -6, and – 7 of the RNA to
keep the RNA-DNA hybrid together.
Structural Basis of Nucleotide Selection
■ Moving through the entry pore toward the active site of RNA
polymerase II, incoming nucleotide first encounters the E (entry) site
* E site is inverted relative to its position in the A site (active) where
phosphodiester bonds form
* E and A sites partially overlap
* Rotation of nucleotide between E and A sites may play a role in base
and sugar specificity
Structural Basis of Nucleotide Selection
(cont…)
■ Two metal ions (Mg2+ or Mn2+ are present at the active site
* One is permanently bound to the enzyme
* The other enters the active site complexed to the incoming
nucleotide.
The Role of Rpb4 and Rpb7
■ TATA box
* Found on the non-template strand
* Very similar to the prokaryotic -10 box
* There are frequently TATA-less promoters
- Housekeeping genes that are constitutively active in nearly all cells
as they control common biochemical pathways
- Developmentally regulated genes.
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