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The C-terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD) plays a key role in transcription initiation at many activator-dependent promoters. This domain is connected to the N-terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on αCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the α linker decrease CRP-dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP-dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker-modified α subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP-dependent promoters. [ABSTRACT FROM AUTHOR]

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRPdependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C-terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase a subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD 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 20310 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between a subunits and CRP, but do not affect DNA binding by a subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in &lpha;CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD-CRP interaction in Class I CRP-dependent transcription. [ABSTRACT FROM AUTHOR]

Living organisms employ endogenous negative feedback loops to maintain homeostasis despite environmental fluctuations. A pressing open challenge in Synthetic Biology is to design and implement synthetic circuits to control host cells' behavior, in order to regulate and maintain desired conditions. To cope with the high degree of circuit complexity required to accomplish this task and the intrinsic modularity of classical control schemes, we suggest the implementation of synthetic endogenous feedback loops across more than one cell population. The distribution of the sensing, computation, and actuation functions required to achieve regulation across different cell populations within a consortium allows the genetic engineering in a particular cell to be reduced, increases the robustness, and makes it possible to reuse the synthesized modules for different control applications. Here, we analyze, in-silico, the design of a synthetic feedback controller implemented across two cell populations in a consortium. We study the effects of distributing the various functions required to build a control system across two populations, prove the robustness and modularity of the strategy described, and provide a computational proof-of-concept of its feasibility.