GUEST COMMENTARY General Pathway for Turning on Promoters Transcribed by RNA Polymerases Containing Alternative Factors

Signal coordinating a response by multiple RNAP holoenzymes. In Escherichia coli, the vast majority of transcription results from the RNA polymerase (RNAP) holoenzyme containing 70 (E ). However, there are six other factors that each recognize other sets of promoters, often united by common function. 32 ( ) controls heat shock promoters, 54 ( ) controls promoters for nitrogen assimilation (as well as some other promoters), S turns on stationary-phase (and other) promoters, F is used for flagellum-related functions, fecI is used for promoters involved in iron transport, and E controls responses to extracytoplasmic stresses. While this may seem like a lot of factors for core RNAP to keep track of, E. coli is actually fairly conservative in its use of alternative factors. For example, Streptomyces coelicolor employs 60 different factors, the majority of which are of the E class. In this issue of the Journal of Bacteriology, Costanzo and Ades (11) examine transcription from two E. coli promoters that turn on as cells approach stationary phase. Even though the promoters are induced at a time characteristic of promoters transcribed by E , they use E E and not E . E -dependent promoters typically are induced by disruptions in the cell envelope (triggered by the misfolding of outer membrane porins, for example) and transcribe mRNAs encoding periplasmic folding catalysts. However, the pathway described by Costanzo and Ades involves the nutritional-stress signal ppGpp and is completely different from the pathway described previously for inducing E -dependent promoters. The major pathway controlling -dependent transcription utilizes a dedicated antifactor, RseA. RseA sequesters E at the inner membrane, preventing it from binding to core RNAP when the cell needs to keep E -dependent promoters inactive. Transcription inhibition is reversed by sequential proteolysis of RseA by two inner membrane proteases, DegS and YaeL (RseP). Regulation by RseA in response to periplasmic stresses has been reviewed recently (1, 2), the genes transcribed by E E (the E regulon) have been identified experimentally and bioinformatically (25), and the X-ray structure of the -RseA complex has been determined (7), bringing an understanding of the sequestration mechanism to the atomic level. Costanzo and Ades (11) show that induction of E dependent promoters as cells go into stationary phase occurs in cells lacking rseA but that it is defective in cells lacking relA and spoT (which code for the two ppGpp synthases) or dksA (a cofactor needed for ppGpp function). ppGpp (guanosine 5 -diphosphate, 3 -diphosphate) is a small molecule long known to be generated in response to depletion of nutrients (e.g., amino acids or carbon) (8, 9). In addition to the E -dependent promoters discussed above, recent studies have shown that some E and E -dependent promoters are also induced by ppGpp/DksA (17, 18, 19). Therefore, Costanzo and Ades (11) suggest that ppGpp and DksA provide a mechanism for activating alternative factors to provide a coordinated response to nutrient depletion. In a sense, this response puts the bacterium on “alert,” preloading it with stress response proteins and the transcriptional potential to counteract the stresses that lie ahead. These results imply that several very large networks of promoters are controlled by a signal whose reach was already known to be very extensive (see also references 20 and 26). Mechanism of ppGpp action on E -dependent promoters. ppGpp was discovered almost 40 years ago, and over the years it has become clear that it induces the collection of responses to starvation known as the stringent response (9). Among these responses are inhibition of stable RNA promoters and activation of certain promoters for amino acid biosynthesis. ppGpp inhibits rRNA promoters to some extent in a pure in vitro system lacking proteins other than RNAP (5), demonstrating that ppGpp works directly on the transcription machinery itself. ppGpp also increases the overall dissociation rate of RNAP from all promoters (5). Since ppGpp is not a sequence-specific DNA-binding protein, its promoter-specific effects on transcription require an alternative explanation. One model is that ppGpp binds to RNAP at all promoters, increasing the decay of the complex directly but inhibiting transcription from only those promoters (e.g., rRNA promoters) where this kinetic step is rate-limiting for transcription. In support of this hypothesis, mutations have been identified in rpoB, rpoC, and rpoD (coding for the , , and 70 subunits of RNAP, respectively) that increase the dissociation rate of the complex, thereby mimicking the effects of ppGpp (4, 16, 27) and bypassing the requirement for relA and spoT. Furthermore, promoter mutations that increase the lifetime of the complex lead to loss of the effects of ppGpp on rRNA transcription in vitro and in vivo (5, 15). Investigators originally were unable to demonstrate positive effects of ppGpp in a pure system in vitro (4, 10). Since rRNA promoters employ a large fraction of RNAP in cells growing at * Corresponding author. Mailing address: University of Wisconsin, Department of Bacteriology, 420 Henry Mall, Madison, WI 53706. Phone: (608) 262-9813. Fax: (608) 262-9865. E-mail: rgourse@bact .wisc.edu.