Promoter recognition in eubacteria is carried out by the initiation factor sigma, which binds RNA polymerase and initiates transcription. Cells have one housekeeping factor and a variable number of alternative sigma factors that possess different promoter-recognition properties. The cell can choose from its repertoire of sigmas to alter its transcriptional program in response to stress. Recent structural information illuminates the process of initiation and also shows that the two key sigma domains are structurally conserved, even among diverse family members. We use the sigma repertoire of Escherichia coli, Bacillus subtilis, Streptomyces coelicolor, and cyanobacteria to illustrate the different strategies utilized to organize transcriptional space using multiple sigma factors.

Multiple sigma subunits and the partitioning of bacterial transcription space.

https://userpages.umbc.edu/~wolf/biol434/busbyebrightRev.1999.pdf

Transcription activation by catabolite activator protein (CAP).

In bacteria, the binding of a single protein, the initiation factor sigma, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A resolution. In the structure, two amino-terminal domains of the sigma subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of sigma is near the outlet of the RNA-exit channel, about 57 A from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.

Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution

An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

Precise and reliable gene expression via standard transcription and translation initiation elements

Bacterial sigma (sigma) factors are an essential component of RNA polymerase and determine promoter selectivity. The substitution of one sigma factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent. As a class, alternative sigma factors play key roles in coordinating gene transcription during various stress responses and during morphological development. The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that are quite divergent in sequence relative to most other sigma factors. Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors and these regulators often outnumber all other types of sigma factor combined. Examples include Bacillus subtilis (7 ECF sigma factors), Mycobacterium tuberculosis (10), Caulobacter crescentus (13), Pseudomonas aeruginosa (approximately 19), and Streptomyces coelicolor (approximately 50). The roles and mechanisms of regulation for these various ECF sigma factors are largely unknown, but significant progress has been made in selected systems. As a general trend, most ECF sigma factors are cotranscribed with one or more negative regulators. Often, these include a transmembrane protein functioning as an anti-sigma factor that binds, and inhibits, the cognate sigma factor. Upon receiving a stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to stimulate transcription. In many ways, these anti-sigma:sigma pairs are analogous to the more familiar two-component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA-binding response regulator. Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane. Here, I review current knowledge of some of the better characterized ECF sigma factors, discuss the variety of experimental approaches that have proven productive in defining the roles of ECF sigma factors, and present some unifying themes that are beginning to emerge as more systems are studied.

J. Tupy

Papers

The functional and regulatory roles of sigma factors in transcription.