RNA Management During T7 Infection.

Post-transcriptional regulation (PTR) determines the fate of RNA in the cell and represents an important control point in the flow of genetic information and thus underpins many, if not all, aspects of cell function. Host takeover by phages through misappropriation of the bacterial transcription machinery is a relatively advanced area of research. However, several phages encode small regulatory RNAs, which are major mediators of PTR, and produce specific proteins to manipulate bacterial enzymes involved in RNA degradation.1-4 However, PTR during phage development still represents an understudied area of phage-bacteria interaction biology. In this study, we discuss the potential role PTR could play in determining the fate of RNA during the lifecycle of the prototypic phage T7 in Escherichia coli.

Xenogeneic Regulation of the Bacterial Transcription Machinery.

The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect bacteria, bacteriophages (phages), are no exception and have evolved sophisticated ways to control essential biosynthetic machineries of their bacterial prey to benefit phage development. The xenogeneic regulation of bacterial cell function is a poorly understood area of bacteriology. The activity of the bacterial transcription machinery, the RNA polymerase (RNAP), is often regulated by a variety of mechanisms involving small phage-encoded proteins. In this review, we provide a brief overview of known phage proteins that interact with the bacterial RNAP and compare how two prototypical phages of Escherichia coli, T4 and T7, use small proteins to "puppeteer" the bacterial RNAP to ensure a successful infection.

T7 phage factor required for managing RpoS in Escherichia coli.

T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.

A Rapid Colorimetric Method to Visualize Protein Interactions.

As key molecules in most biological pathways, proteins physically contact one or more biomolecules in a highly specific manner. Several driving forces (i.e., electrostatic and hydrophobic) facilitate such interactions and a variety of methods have been developed to monitor these processes both in vivo and in vitro. In this work, a new method is reported for the detection of protein interactions by visualizing a color change of a cyanine compound, a supramolecule complex of 3,3-di-(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt (MTC). Nuclear magnetic resonance (NMR) studies suggest that the hydrophobic nature of the protein surfaces drives MTC into different types of aggregates with distinct colors. When proteins interact with other biomolecules, the hydrophobic surface of the complex differs, resulting in a shift in the form of MTC aggregation, which results in a color change. As a result, this in vitro method has the potential to become a rapid tool for the confirmation of protein-biomolecule interactions, without the requirements for sophisticated instrumentation or approaches.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA.

Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping σ-factor, σ(A). The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ(A) as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.

The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.