can also influence promoter function without the need to contact RNA polymerase. The leuV operon consists of three genes that encode three of the four tRNA1Leu isoreceptors and its promoter is under the positive control of FIS. The single binding site for the FIS protein upstream of the leuV promoter is located in a DNA segment that is prone to becoming single‐stranded under the torsional stress imposed by negative supercoiling. This phenomenon is known as supercoiling‐induced DNA duplex destabilisation, SIDD (Benham 1992, 1993). Binding of the FIS protein to its site within the SIDD element displaces the tendency towards duplex destabilisation to the nearest susceptible site, in this case, the leuV promoter – assisting in the formation there of an open transcription complex (Opel et al. 2004). This mechanism is not peculiar to FIS and has been demonstrated for the IHF NAP too (Sheridan et al. 1998). It is likely to be used at many other promoters and represents an under‐researched aspect of the link between NAP binding, DNA topology, and promoter activation.
FIS has also been shown to create a nucleoprotein complex at promoters with a series of FIS‐binding sites that stabilise the topological state of the DNA in ways that favour transcription initiation (Rochman et al. 2004). Many of these promoters express genes that encode components of the translational apparatus, such as ribosomal proteins, tRNA, and rRNA (Champagne and Lapointe 1998; Newlands et al. 1992; Nilsson et al. 1990). Increased translation capacity is necessary to support rapid bacterial growth, so the stimulatory role of FIS during the lag‐to‐log phase of the growth cycle is important. Consistent with this is the observation that while mutants that lack the FIS protein remain viable, they display reduced competitive fitness when grown in co‐culture with their otherwise isogenic wild‐type parent (Schneider et al. 1997).
1.39 FIS and the Stringent Response
Stable RNA (tRNA and rRNA) genes that are stimulated by FIS are subject to control by the stringent response (Condon et al. 1995b; Potrykus and Cashel 2008). Here, an intracellular signal known as an ‘alarmone’ interferes with the ability of RNA polymerase to transcribe a subset of genes, including the stable RNA genes. The alarmone is guanosine tetraphosphate (ppGpp) or pentaphosphate (pppGpp) and it is synthesised in response to a build‐up of uncharged tRNA molecules and the interaction of the RelA protein with stalled ribosomes (Brown et al. 2016; Hauryliuk et al. 2015; Richter 1976) (see Section 6.18 for a more complete description of the stringent response). This accumulation results from a shortage of amino acids to charge the tRNAs and is an indication that the translational capacity of the cell exceeds demand. Hence the feedback loop that shuts down the transcription of genes involved in the production of ribosomes and other parts of the translational apparatus. The stringent response also affects DNA synthesis and mRNA translation both negatively and directly, while stimulating the transcription of genes outside the stringently regulated group (Ferullo and Lovett 2008; Haugen et al. 2006; Paul et al. 2005). What distinguishes the members of the two groups? One important factor is the possession by stringently regulated promoters of a discriminator sequence consisting of a G+C‐rich DNA between its −10 and +1 elements (Figure 1.19) (Lamond and Travers 1985; Mizushima‐Sugano and Kaziro 1985; Travers 1980; Travers et al. 1986; Zacharias et al. 1989). The discriminator is an effective barrier to open complex formation, possibly due to the extra hydrogen bonding between DNA strands consisting of G+C‐rich sequences. Negative supercoiling of the DNA has a stimulatory effect on the promoters of stable RNA genes and this may assist with the melting of the recalcitrant discriminators when negative supercoiling is available (Schneider et al. 2000). However, in bacteria experiencing low metabolic flux (e.g. those in lag phase or stationary phase) this stimulatory influence is absent and the resulting relaxation of the DNA template, combined with the negative influences of the (p)ppGpp alarmone and the DksA protein, cooperate to repress transcription of stable RNA genes (Potrykus and Cashel 2008; Schneider et al. 2000). Genes subject to stimulation by (p)ppGpp and DksA also possess a discriminator, but in these cases this element is an A+T‐rich DNA sequence (Figure 1.19) (Gummesson et al. 2013).
Figure 1.19 The multifaceted stringent response. A summary is shown of the processes that are inhibited or enabled by the alarmone (p)ppGpp. (a) (p)ppGpp and DksA affect a stringently regulated promoter that contains a G+C‐rich discriminator sequence negatively. Genes encoding rRNA or tRNA are in this category. (b) In contrast, (p)ppGpp and DksA affect a promoter with an A+T‐rich discriminator positively. Genes involved in amino acids biosynthesis are in this category. (c) The (p)ppGpp alarmone biases the selection of sigma factors by RNA polymerase away the RpoD housekeeping sigma factor and towards sigma factors that are required for various stress responses. (d) The initiation of chromosome replication is inhibited by (p)ppGpp. (e) Translation initiation and translation elongation are affected negatively because (p)ppGpp has an inhibitory influence on Initiation Factor 2 (IF2) and on the translation elongation factor, EF, respectively.
1.40 FIS and DNA Topology
The involvement of FIS in SIDD‐based regulatory mechanisms has already been described. The promoter of the dusB‐fis operon is subject to transcriptional stimulation by DNA negative supercoiling (Schneider et al. 2000) in addition to being auto‐repressed by FIS and controlled negatively by the stringent response (Ninnemann et al. 1992). At a global level, the FIS protein is intimately associated with the general management of DNA topology in the bacterial cell. It represses the transcription of the gyrA and gyrB genes in E. coli (Schneider et al. 1999) and Salmonella (Keane and Dorman 2003) and has a complicated relationship with the promoters of the topA gene, where its influence is conditional on factors such as oxidative stress (Weinstein‐Fischer and Altuvia 2007). Although E. coli and Salmonella have distinct DNA supercoiling set points, with Salmonella DNA being more relaxed than in E. coli (Champion and Higgins 2007), this distinction is dependent on the presence of FIS (Cameron et al. 2011). Thus, the pattern of expression of the topoisomerases responsible for negative supercoiling (DNA gyrase) and relaxation (Topo I) of DNA is modulated by FIS. The activities of these topoisomerases is also affected by FIS because the protein influences their access to DNA by binding to it: since FIS prefers to bind to DNA with intermediate levels of negative supercoiling it acts to preserve this topological form (Schneider et al. 1997; Cameron and Dorman 2012).
In order to exert its influence on DNA topology, FIS must be present in the cell. This restricts its influence to the early stages of exponential growth when it is most abundant (Schneider et al. 1997). An exception has been discovered in bacteria growing under micro‐aerobic conditions: here FIS levels are sustained into the stationary phase of growth (Cameron et al. 2013; O Cróinín and Dorman 2007). This may be of special significance in environments such as the mammalian gut epithelial surface where FIS‐dependent gene expression is required for colonisation and invasion (Falconi et al. 2001; Kelly et al. 2004; Prosseda et al. 2004; Rossiter et al. 2015).
1.41 Ferritin‐Like Dps and the Curved‐DNA‐binding Protein CbpA
While FIS is associated with the early stages of rapid exponential growth, the Dps (DNA‐binding protein from starved cells) and CbpA proteins exhibit the polar opposite expression pattern and are seen predominantly in stationary phase (Ali Azam and Ishihama 1999). Both are NAPs and Dps has been studied in the most detail. CbpA expression is prevented during exponential growth by the FIS protein. FIS, which is abundant in this period of the growth cycle, binds and represses the activity of an RpoD‐dependent promoter that is located in a gene (yccE) adjacent to cbpA that is partly responsible for cbpA transcription in stationary phase. A second promoter immediately upstream of cbpA depends on RpoS, a sigma factor that is only available
