Abstract
Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoHI and RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the −35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several bases in the predicted −10 element were important for activity with either RpoHII or both RpoH homologs. Genes that are transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.
Highlights
Transcriptional responses to stress are critical to cell growth and survival
The Western blot analysis used to measure levels of these alternative s factors demonstrates that cells ectopically expressing RpoHI and RpoHII contained each protein at levels comparable to those following either heat shock or singlet oxygen stress (Figure 1)
These strains can be used to characterize members of the RpoHI and RpoHII regulons. As controls for this experiment, we measured the abundance of individual RpoH proteins and a control transcription factor (PrrA) [23], which is not known to be dependent on either alternative s factor for its expression, when wild type cells were exposed to either heat or singlet oxygen stress
Summary
Transcriptional responses to stress are critical to cell growth and survival. In bacteria, stress responses are often controlled by alternative s factors that direct RNA polymerase to transcribe promoters different from those recognized by the primary s factor [1,2]. The transcriptional response to heat shock in Escherichia coli uses the alternative s factor s32 to increase synthesis of gene products involved in protein homeostasis or membrane integrity [3]. Proteins related to E. coli s32 are conserved across virtually all proteobacteria. This so-called RpoH family of alternative s factors is characterized by a conserved amino acid sequence (the ‘‘RpoH box’’) that is involved in RNA polymerase interactions [4,5]. RpoH family members possess conserved amino acid sequences in s factor regions 2.4 and 4.2 that interact with promoter sequences situated approximately 210 and 235 base pairs upstream of the transcriptional start sites, respectively [6]
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