BIMM 122 Lecture Notes #11 Dr. Milton Saier

Activation of Transcriptional (Tx) Initiation Complexes

One of the best examples of Tx initiation activation results from studies of the Cyclic AMP Receptor Protein (CRP or CAP). CRP activates more operons in E. coli than any other transcription factor. It controls carbon and energy metabolism at many levels, but particularly by regulating the initial metabolic steps in the utilization of exogenous carbon sources. It influences Tx by several mechanisms, mostly by activation but sometimes by repression. Often it binds to several sites in the promoter region of any one target operon.

In Class I activation, CRP binds upstream of the promoter (at positions -61 to -91 relative to the Tx start site) and also by binding the CTD (RNA polymerase (RNAP), -subunit, C-terminal domain) when bound to the DNA on the same face of the double helix as RNAP. This interaction promotes binding of another region of -CTD to the minor groove, adjacent to and downstream of CRP. -CTD also binds 70, activating RNAP for promoter binding followed by open complex formation.

In Class II activation, CRP binds at position -41, overlapping the -35 promoter region on the same side of the helix as RNAP. Two regions of CRP bind -CTD and -NTD, respectively, while a third region binds 70, all on the same side of the helix. The overlapping binding sites for 70 and CRP prevent full binding to either site, and the DNA stabilizes the interaction by wrapping around the complex.

CRP bends the DNA ~90o and can function by aligning and orienting different parts of the transcriptional initiation complex. Thus, CRP may bind to multiple sites in a single promoter, sometimes cooperatively, sometimes antagonistically (consider gal, glnAp2 and mtl operon activation in E. coli).

Enhancers, especially 54-dependent enhancers, bind to an upstream activating sites (UASs) (positions -80 to -150) that do not overlap the promoter, on the face of the DNA opposite to that of RNAP. Contact is made with 54 via DNA looping, sometimes facilitated by binding of other proteins such as IHF or another nucleoid protein. Without the enhancer, 54 physically blocks the RNAP channel for entry of the DNA template strand into the RNAP active site. Interaction with the enhancer activates its ATPase, brings about a sequential series of conformational changes in the complex, opens the channel, opens the strands at the -12 site and aligns the promoter with the active site within the channel. Thus, while 70 promotes open complex formation without assistance by other proteins, 54 promotes closed complex formation, and the enhancer must open the complex. While 70 primarily activates housekeeping promoters, 54 primarily activates stress-relieving promoters, especially nitrogen (and sometimes carbon) starvation stress in E. coli.

Enhancement was originally discovered in eukaryotes and was thought to be characteristic of these organisms, even being lacking in prokaryotes. We now believe that in eukaryotes, transcriptional initiation control evolved from the bacterial 54-dependent mechanism while 70 was lost during eukaryotic evolution.

References:Ghosh, T., D. Bose, and X. Zhang (2010) Mechanisms for activating bacterial RNA polymerase. FEMS Microbiol. Rev. 34: 611-627. [PMID: 20629756]