Polymerase Alpha Subunit C-terminal Domain Research Articles

Crystal Structure of Bacteriophage λcII and Its DNA Complex

The tetrameric cII protein from bacteriophage lambda activates transcription from the phage promoters P(RE), P(I), and P(AQ) by binding to two direct repeats that flank the promoter -35 element. Here, we present the X-ray crystal structure of cII alone (2.8 A resolution) and in complex with its DNA operator from P(RE) (1.7 A resolution). The structures provide a basis for modeling of the activation complex with the RNA polymerase holoenzyme, and point to the key role for the RNA polymerase alpha subunit C-terminal domain (alphaCTD) in cII-dependent activation, which forms a bridge of protein/protein interactions between cII and the RNA polymerase sigma subunit. The model makes specific predictions for protein/protein interactions between cII and alphaCTD, and between alphaCTD and sigma, which are supported by previous genetic studies.

Interdependence of Activation at rhaSR by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR

The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of L-rhamnose. beta-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at -111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase alpha-subunit C-terminal domain (alpha-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of alpha (alpha-Delta235), and alanine substitutions throughout alpha-CTD. We found that DNA-contacting residues in alpha-CTD are required for full activation, and for simplicity, we discuss alpha-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and alpha-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an alpha-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and alpha-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the alpha-CTD-CRP and alpha-CTD-RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, alpha-CTD, and RhaR.

Synergistic transcription activation: a dual role for CRP in the activation of an Escherichia coli promoter depending on MalT and CRP.

Activation of the Escherichia coli malEp promoter relies on the formation of a higher order structure involving cooperative binding of MalT to promoter-proximal and promoter-distal sites as well as CRP binding to three sites located in between. MalT is the primary activator and one function of CRP is to facilitate cooperative binding of MalT to its cognate sites by bending the intervening DNA. It is shown here that CRP also participates directly in malEp activation. This function is carried out by the molecule of CRP bound to the CRP site centered at -139.5 (CRP site 3). This molecule of CRP recruits RNA polymerase by promoting the binding of the RNA polymerase alpha subunit C-terminal domain (alphaCTD) to DNA immediately downstream from CRP site 3, via a contact between alphaCTD and activating region I of CRP. The action of MalT and CRP at malEp hence provides the example of a novel and complex mechanism for transcriptional synergy in prokaryotes whereby one activator both helps the primary activator to form a productive complex with promoter DNA and interacts directly with RNA polymerase holoenzyme.

Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.