Group 3 sigma factors in the marine cyanobacterium Synechococcus sp. strain PCC 7002 are required for growth at low temperature.

Three genes, sigF, sigG and sigH, encoding group 3 sigma factors have been cloned and characterized in the marine cyanobacterium Synechococcus sp. strain PCC 7002. The sigF gene product was similar to sigma factors involved in general stress response and sporulation in other organisms, and the sigG and sigH gene products were similar to extracytoplasmic function (ECF) sigma factors. The sigG and sigH genes were associated with the putative regulatory genes and the sizes of transcripts for sigG and sigH genes were large enough to be cotranscribed with the associated downstream genes. The sigG downstream gene was designated sapG (sigG-associated protein), and yeast two-hybrid analysis demonstrated that SigG and SapG interact when produced in yeast cells. Null mutants of these three group 3 sigma factor genes were created by interposon mutagenesis. The growth of the sigF mutant strain was much slower than the wild-type strain at 15 degrees C, although the growth rates at 22 degrees C and 38 degrees C were identical to those of the wild-type strain. The sigG mutant could not grow continuously at 22 degrees C, and no growth occurred at 15 degrees C. Since SigG and SapG interact in yeast cells and the sigG and sapG mutants showed a similar growth phenotype, SapG is likely to be a regulatory protein for SigG involved in the same pathway in transcriptional regulation in this cyanobacterium.

A hydrophobic patch on the flap-tip helix of E.coli RNA polymerase mediates sigma(70) region 4 function.

The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel.

Multiple sigma subunits and the partitioning of bacterial transcription space.

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.

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.

Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting.

We determined the minimal portion of Escherichia coli RNA polymerase (RNAP) holoenzyme able to accomplish promoter melting, the crucial step in transcription initiation that provides RNAP access to the template strand. Upon duplex DNA binding, the N terminus of the beta' subunit (amino acids 1 to 314) and amino acids 94 to 507 of the sigma subunit, together comprising less than one-fifth of RNAP holoenzyme, were able to melt an extended -10 promoter in a reaction remarkably similar to that of authentic holoenzyme. Our results support the model that capture of nontemplate bases extruded from the DNA helix underlies the melting process.

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.

Crystal structure of Escherichia coli sigmaE with the cytoplasmic domain of its anti-sigma RseA.

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

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

Bacterial sigma 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 sigma 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 sigma70-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.

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.