Targets for the MalI repressor at the divergent Escherichia coliK-12malX-malI promoters

Random mutagenesis has been used to identify the target DNA sites for the MalI repressor at the divergent Escherichia coli K-12 malX-malI promoters. The malX promoter is repressed by MalI binding to a DNA site located from position -24 to position -9, upstream of the malX promoter transcript start. The malI promoter is repressed by MalI binding from position +3 to position +18, downstream of the malI transcript start. MalI binding at the malI promoter target is not required for repression of the malX promoter. Similarly, MalI binding at the malX promoter target is not required for repression of the malI. Although the malX and malI promoters are regulated by a single DNA site for cyclic AMP receptor protein, they function independently and each is repressed by MalI binding to a different independent operator site

Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production

The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give controlled expression that is suitable for diverse biotechnology applications

Laboratory strains of Escherichia coli K-12: not such perfect role models after all

Escherichia coli K-12 was originally isolated 100 years ago and since then, it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its apparent pedigree, since its initial isolation, E. coli K-12 has been repeatedly cultured, passaged, and mutagenized, resulting in an organism that carries extensive genetic changes. To understand more about the evolution of this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive lab-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains, leads us to question whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology

The C-terminal domain of the Escherichia coli RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ pM promoter

The bacteriophage λ pM promoter is required for maintenance of the λ prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the λ repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of pM. CI activates pM through cooperative binding to the OR1 and OR2 sites within the OR operator, with the OR2-bound CI dimer making contact with domain 4 of the RNA polymerase σ subunit (σ4). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase α subunit (αCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of pM. We also show that the location of αCTD at the pM promoter changes in the presence of CI. Thus, in the absence of CI, one αCTD is located on the DNA at position −44 relative to the transcription start site, whereas in the presence of CI, αCTD is located at position −54, between the CI-binding sites at OR1 and OR2. These results suggest that contacts between CI and both αCTD and σ are required for efficient CI-dependent activation of pM

The Escherichia coli RNA polymerase α subunit linker: length requirements for transcription activation at CRP-dependent promoters

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

A simple mechanism for co-dependence on two activators at an Escherichia coli promoter

The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence. MelR binds to four sites, centred at positions –42.5, –62.5, –100.5 and –120.5 relative to the melAB transcription start point. When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions –62.5 and –100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase α subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome

Requirement for two copies of RNA polymerase α subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length α subunits, or containing one full-length α subunit and one truncated α subunit lacking the α C-terminal domain (αCTD). At a promoter having a single DNA site for CRP, activation requires only one full-length α subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for αCTD), activation requires only one full-length α subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length α subunits. We conclude that a single copy of αCTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of αCTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites