The a Subunit of E. coli RNA Polymerase

The alpha subunit has two separate domains

• the amino terminal domain is essential for dimerization and assembly of polymerase,
• the carboxy terminal domain is needed for binding to DNA and for communication with many, but not all, transcription factors.

The a-NTD makes contact with the b and b' subunits of RNAP, and is conserved in the 40 kDa subunit of eukaryotic polymerase.

Most RNA polymerase (~60%) is associated with rRNA or tRNA genes.

• This is accomplished by a special sequence upstream of the promoter (-35) and (-10) elements, called the UP element (-57)5'-AAAATTATTTT-3'(-47) (Estrem et al, 1998), which
  • binds a₂ dimers
  • increases occupancy by polymerase by ~10-fold.

• This DNA binding motif is novel.
  • <> It is comprised of
    • 1 "unusual helix" (blue coil in structure model)
    • four normal helixes (red)
  • DNA contacts occur between helix-1 and the loop between helicees 3 and 4.
  • Mutations have revealed that two regions of the domain are most crucial for DNA binding (Gaal et al., 1996):
    • <> residues L262, R265, N268, and C269 define region I (yellow)
    • <> residues G296, K298, and S299 define region II (red).

RNA polymerase also has to communicate with transcription factors.

• The geometry of the polymerase/promoter complex makes this possible.
• Much of the communication between activators and E. coli polymerase is mediated between the CTD of a and these factors.
  • <> For CAP, region 258-265 of the a subunit (red) is important, with residue 261 being most crucial (red spacefill).
    • Allele specific phenotypes indicate that this region is distinct from that which "sees" UP elements;
    • At these promoters, called Class I CAP-dependent promoters, CAP binds at -62, -73, -83 or -93, which are helically phased positions relative to the promoter.
    • Only 1 a subunit's CTD need be present in the polymerase to interact with CAP.
    • This flexibility is presumed to rely on a flexible nature for the linker that joins the NTD and CTD of the a subunit.
    • It is believed that this interaction between CAP and the a-CTD substitutes for an UP element at the CAP activated promoters, increasing K_b.

Other genetic evidence indicates that the a-CTD communicates with additional factors (see Ebright and Busby, 1995).

• <> P2-Ogr and P4-d (yellow spacefill)
• <> FNR (blue spacefill)
• <> OmpR (green spacefill)
• <> AraC, CysB, MelR (pink spacefill)
• <> ADP-ribosylation occurs at Arg265 (cyan spacefill), letting phage T4 take over the polymerase.

And the a subunit can interact with CAP via an entirely different mechanism (Ebright et al, 1997).

• The a subunit inhibits polymerase binding at CAP Dependent, Type II promoters which lack -34 promoter elements. Instead, these promoters have a binding site for CAP centered at -42.
• The inhibition of polymerase binding by the a subunit is overcome by a positive interaction between part of CAP (activating region AR1 - amino acids 156-164) and the CTD of the a subunit (residues 265, 271, 285-288, and 317; Savery et al; 1998)
  • These are clearly two distinct parts of the a-CTD that interact with distinct parts of CAP protein under different promoter configurations.
    • <> red spacefill - the part of the a subunit that interacts with CAP at CAP Depentent, Class II promoters.
    • <> cyan spacefill - the part of the a subunit that interacts with CAP at CAP-Dependent, Class I promoters as described above).
• This interaction is then followed by isomerization of closed to open complex, mediated by CAP activating region 2 (amino acids 19, 21, 96 and 101) interacting with the a-NTD.