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Journal of Bacteriology, July 2006, p. 4589-4591, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.00499-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

GUEST COMMENTARY

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

Richard L. Gourse,^* Wilma Ross, and Steven T. Rutherford

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

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 (E⁷⁰). However, there are six other factors that each recognize other sets of promoters, often united by common function. ³² (^H) controls heat shock promoters, ⁵⁴ (^N) 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^S, they use E^E and not E^S. 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^E-dependent promoters.

The major pathway controlling ^E-dependent transcription utilizes a dedicated anti- factor, RseA. RseA sequesters ^E at the inner membrane, preventing it from binding to core RNAP when the cell needs to keep E^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 ^E-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^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^E-dependent promoters discussed above, recent studies have shown that some E^S- and E⁵⁴-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 E⁷⁰-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 ß, ß', 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 moderate-to-high growth rates, this led to the proposal that positive control by ppGpp might be passive (i.e., indirect), resulting from an increase in the availability of RNAP that would derive from inhibition of rRNA transcription. Several lines of evidence supported this model. For example, the RNAP mutants described above that simulated the negative effects of ppGpp also stimulated promoters (i.e., the E⁷⁰-dependent amino acid biosynthetic promoters mentioned above) that are positively regulated by ppGpp, but only in vivo, not in vitro. Furthermore, gross overproduction of RNA or underproduction of RNAP preferentially decreased transcription from the amino acid promoters that are positively regulated by ppGpp. Finally, these promoters were demonstrated to have kinetic characteristics that fit the predictions of the indirect model: they form relatively long-lived complexes with RNAP (and are thus insensitive to the negative effects of ppGpp), and they have very weak binding constants for RNAP (and thus require high concentrations of RNAP for transcription).

The identification of DksA as a cofactor for ppGpp function resolved discrepancies in the effects of ppGpp on transcription observed in vivo versus in vitro (22, 23). dksA mutants were shown to be defective in both negative and positive control in vivo, and when purified DksA was included in transcription reactions, large negative and positive effects of ppGpp were observed in vitro. Although DksA plays a critical role in both direct negative and direct positive responses to ppGpp, the data described above suggest that not all of the effect of ppGpp/DksA on positive control is direct; positive control also results from indirect effects of ppGpp/DksA on transcription (from reducing the use of RNAP elsewhere and thereby increasing the effective RNAP concentration).

ppGpp and DksA have been proposed to interact with each other in the secondary channel of RNA polymerase (3, 24). This model requires confirmation, but even if correct, how this accounts for the effects of ppGpp/DksA on transcription remains to be determined.

Effects of ppGpp/DksA on transcription by alternative holoenzymes.

The mechanism by which ppGpp/DksA redistributes RNAP to E^E-dependent promoters has not yet been determined. There is considerable evidence for competition among factors for core RNAP in vivo. For example, superinduction of some E⁷⁰-dependent promoters is observed in rpoS mutants, and overexpression of ⁷⁰ reduces E^S-dependent transcription (12). Since ppGpp/DksA is required for, or increases, transcription from some promoters recognized by E³², E^S, E⁵⁴, and E^E (6, 11, 14, 17, 18, 19), one model is that ppGpp/DksA directly affects this competition, favoring assembly of at least some alternative holoenzymes (model 1; reviewed in reference 21). However, there is little evidence that ppGpp/DksA directly increases either the dissociation of ⁷⁰ from core RNAP or the binding of alternative factors to core RNAP. At least two other models could also account for the effects of ppGpp/DksA on promoters using alternative holoenzymes. Some of these promoters could have kinetic properties that lead to their direct stimulation by ppGpp/DksA (model 2), as described above for certain E⁷⁰-dependent amino acid promoters, or the observed in vivo stimulation by ppGpp/DksA could be indirect, ascribable to an increase in the availability of RNAP (model 3).

The absolute concentrations of core RNAP and of the different factors, as well as the level of nonspecific binding of RNAP to DNA, could influence the effects of ppGpp/DksA on transcription by the alternative holoenzymes in vivo. These key parameters have been reexamined recently (13), and it is clear that the level of nonspecific DNA binding by RNAP, the amount of ^E, and the ratio of to core are all higher than proposed previously. Although these results do not resolve how ppGpp/DksA favors transcription by the alternative holoenzymes, it appears that appropriate conditions exist for ppGpp/DksA to play a role in the redistribution of RNAP, especially to weak promoters recognized by alternative holoenzymes.

A recent report from the Shingler laboratory addresses models for the effects of ppGpp/DksA on the Pseudomonas putida Po promoter. This promoter is recognized not only by P. putida E⁵⁴ but also by E. coli E⁵⁴, and its activity increases in a relA spoT- and dksA-dependent manner in vivo (6, 19). However, stimulation of this promoter by ppGpp/DksA was not observed in vitro, either in the presence or the absence of competing transcription by other holoenzymes. Since stimulation of some E⁷⁰-dependent amino acid promoters by ppGpp/DksA was observed by these investigators (6), they suggest that the effects of ppGpp/DksA on E⁵⁴-dependent transcription might all be indirect, resulting only from effects of ppGpp/DksA on increasing the availability of RNAP. Similar tests of the mechanisms underlying the ppGpp/DksA requirement for E³², E^S, and E^E-dependent promoters would be helpful in distinguishing whether their positive control by ppGpp/DksA is direct or indirect.

Future prospects.

In summary, the idea that the induction of ppGpp provides a general mechanism for coordinately activating promoters that use alternative factors is an exciting one. Current data now support this idea for promoters using four different alternative holoenzymes (E^S, E⁵⁴, E³², and E^E). Hopefully effects of ppGpp/DksA on transcription by the other two alternative holoenzymes will be tested soon. Additional studies will be required to distinguish between the potential mechanisms described above. Definitive measurements of association and dissociation constants for the different factors with core RNAP are required (individually and together in the same reaction), and the effects of ppGpp/DksA on these constants are needed to determine whether ppGpp/DksA directly alters competition between factors for core RNAP (model 1 above). Furthermore, the three different mechanisms for ppGpp/DksA action described above are not mutually exclusive. Both direct and indirect mechanisms play a part in the positive control of certain E⁷⁰-dependent promoters by ppGpp/DksA (models 2 and 3 above [4, 23]). Effects of ppGpp/DksA do not occur in a vacuum; other transcription factors (e.g., 6S RNA, anti- factors, and classical activators and repressors) could also contribute to the redistribution of holoenzymes on cellular promoters. Finally, just as different E⁷⁰-dependent promoters have different kinetic characteristics, resulting in different responses to ppGpp/DksA, this could also be true for different promoters in the same alternative holoenzyme class. While the promoter population recognized by an alternative holoenzyme is not likely to be as diverse kinetically as the population recognized by E⁷⁰ (see reference 25 for a discussion of this issue), it would be surprising if evolution had never taken advantage of the available diversity in promoter sequence to distinguish between promoters recognized by a single alternative holoenzyme.

ACKNOWLEDGMENTS

Work in our laboratory is supported by NIH grant GM37048.

We thank S. Ades and V. Shingler for comments on the manuscript.

 
* 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{at}bact.wisc.edu.

FOOTNOTES

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

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Journal of Bacteriology, July 2006, p. 4589-4591, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.00499-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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