Tanja M. Gruber

The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium

The complete genome of the green-sulfur eubacterium Chlorobium tepidum TLS was determined to be a single circular chromosome of 2,154,946 bp. This represents the first genome sequence from the phylum Chlorobia, whose members perform anoxygenic photosynthesis by the reductive tricarboxylic acid cycle. Genome comparisons have identified genes in C. tepidum that are highly conserved among photosynthetic species. Many of these have no assigned function and may play novel roles in photosynthesis or photobiology. Phylogenomic analysis reveals likely duplications of genes involved in biosynthetic pathways for photosynthesis and the metabolism of sulfur and nitrogen as well as strong similarities between metabolic processes in C. tepidum and many Archaeal species.

Crystal Structure of Escherichia coli σE with the Cytoplasmic Domain of Its Anti-σ RseA

Abstract The σ factors are the key regulators of bacterial transcription. ECF (extracytoplasmic function) σs are the largest and most divergent group of σ 70 family members. ECF σs are normally sequestered in an inactive complex by their specific anti-σ factor, which often spans the inner membrane. Here, we determined the 2 A resolution crystal structure of the Escherichia coli ECF σ factor σ E in an inhibitory complex with the cytoplasmic domain of its anti-σ, RseA. Despite extensive sequence variability, the two major domains of σ E are virtually identical in structure to the corresponding domains of other σ 70 family members. In combination with a model of the σ E holoenzyme and biochemical data, the structure reveals that RseA functions by sterically occluding the two primary binding determinants on σ E for core RNA polymerase.

Views of transcription initiation.

Initiation of transcription is the first step in gene expression and a major point of regulation. Recent structural studies reveal the nature of the initiating complex and suggest new ways of accomplishing the processes required for initiation.

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Bacterial σ factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the −10 and −35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of σ is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the −35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a σ70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.

A Coiled-Coil from the RNA Polymerase β′ Subunit Allosterically Induces Selective Nontemplate Strand Binding by σ70

Abstract For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the −10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the β′ subunit (aa 262–309) induces σ 70 to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the β′ coiled-coil and region 2.2 of σ 70 promotes an allosteric transition that allows σ 70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ 70 , nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Binding of the Initiation Factor σ70 to Core RNA Polymerase Is a Multistep Process

Abstract The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and σ 70 , the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between σ 70 and specific domains of RNA polymerase and provide evidence that σ 70 and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of σ 70 with core RNA polymerase is associated with progression through the process of transcription initiation.

Expression of two alternative sigma factors of Synechococcus sp. strain PCC 7002 is modulated by carbon and nitrogen stress

The sigB and sigC genes, encoding two alternative sigma factors of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigB and sigC genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigB and SigC are group 2 sigma factors. Starvation for either nitrogen or carbon caused an increase in sigB mRNA levels. Transcripts for the sigC gene initially increased but then decreased during nitrogen and carbon starvation. The SigC protein could not be identified in cyanobacterial extracts using antisera to Synechococcus sp. PCC 7002 SigA or RpoD from Bacillus subtilis. The ratio of the principal vegetative sigma factor, SigA, to SigB decreased during either nitrogen starvation or carbon starvation, and the levels of SigB also increased in the sigC mutant strain. These results imply that SigB and SigC play roles in modifying transcription in response to changes in carbon and nitrogen availability in this cyanobacterium.

Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus

The group 1 and group 2 σ70-type sigma factors of the green sulfur bacterium Chlorobium tepidum and of the green nonsulfur bacterium Chloroflexus aurantiacus were cloned and characterized. Cb. tepidum was found to contain one σ70-type sigma factor; the expression of the gene was analyzed by Northern blot hybridization and primer-extension mapping. Cf. aurantiacus has genes encoding four sigma factors of groups 1 and 2. The expression of these genes was examined in cells grown aerobically and anaerobically. The sigC gene was expressed at approximately equal levels under both conditions, resulting in its designation as the group 1 sigma factor of this organism. The only other detectable transcripts arose from the sigB gene, which was expressed at higher levels during aerobic growth. A phylogenetic tree was obtained using the group 1 sigma factors of Cb. tepidum, Cf. aurantiacus, and diverse eubacteria as the molecular marker. The resulting phylogenetic tree shows that Cb. tepidum and Cf. aurantiacus are related to each other and to the cyanobacteria. The relationship of the group 2 sigma factors of Cf. aurantiacus and the cyanobacteria was more specifically examined phylogenetically. The group 2 sigma factors of Cf. aurantiacus probably arose by gene duplication events after the split of the green nonsulfur bacteria from other photosynthetic eubacteria.

Assay of Escherichia coli RNA Polymerase: Sigma–Core Interactions

Publisher Summary This chapter reviews that the sigma subunit (σ) of prokaryotic RNA polymerase confers promoter specificity to the enzyme. It not only recognizes the promoter DNA, but also triggers a series of structural transitions required for initiation. Genetic and biochemical methods have shown that the σ subunit can form contacts with core RNA polymerase along the length of the protein. The chapter reviews the approach that is useful to study interactions of regions, not resolved in the crystal structure, along with the determination of interactions that occur at an initial stage versus interactions that occur subsequent to conformational changes of the two binding partners. Domains masking interaction sites within the protein can also be identified. Furthermore, interactions of core with σ factors other than housekeeping σ factors is also investigated and compared. This approach, in principle, is used to examine any protein–protein interaction.

An Overview of RNA Polymerase Sigma Factors in Phototrophs

Sigma factors are dissociable subunits that confer promoter specificity on eubacte-rial core RNA polymerase and are required for transcription initiation. Two major families of sigma factors occur in eubacteria: the a70 (RpoD) family [1,2]) and the a54 (RpoN) family [3]; which are both named after the originally identified Escherichia coli proteins. Based upon sequence comparisons and functional considerations, the σ70 family has been divided into three groups [1]. The primary sigma factors, which are essential for cell viability, comprise group 1. Group 2 includes alternative sigma factors that are highly similar in sequence to the respective group 1 members, whereas group 3 sigma factors are alternative sigma factors that vary more significantly in sequence from the other two groups and include functional groupings such as heat shock and sporulation sigma factors. Sigma factors of groups 2 and 3 are not required for cell viability. Group 1 and group 2 members are related sufficiently closely that cross-hybridization is easily detected in genomic Southern analyses.