the regulation of transcription initiation in bacteria...that is, the frequency of transcription...

Ann. Rev. Genet. 1985. 19:35547Copyright © 1985 by Annual Reviews Inc. All rights reserved

THE REGULATION OFTRANSCRIPTION INITIATIONBACTERIA

IN

William S. Reznikoff I, Deborah A. Siegele 2, Deborah W.Cowingz, and Carol A. Gross2

Departments of Biochemistry~ and Bacteriology2, College of Agricultural and LifeSciences, University of Wisconsin, Madison, Wisconsin 53706

CONTENTS

INTRODUCTION ..................................................................................... 355PROMOTER STRUCTURE ......................................................................... 356THE REGULATION OF E~TM TRANSCRIPTION INITIATION ............................ 360

Negative Regulation of Transcription Initiation ............................................. 361Positive Regulation of Transcription Initiation .............................................. 363DNA Topology Regulation of Transcription Initiation ...................................... 365DNA Modification and the Regulation of Gene Expression ............................... 366

MODIFICATION OF HOLOENZYME STRUCTURE AND THE REGULATION OFGENE EXPRESSION ................................................................... 366

E. coil ~r7° .......................................................................................... 367The Heat Shock Response and ~r~2 ............................................................. 368The E. coli ntrA (glnF) Protein: Another Sigma Factor ................................... 370B. subtilis Sporulation ........................................................................... 371Sigma Factors in B. subtilis .................................................................... 372Phage-Encoded Sigma Factors ................................................................. 376Conservation of Structure Among Sigma Factors ........................................... 377Modulation of Sigma Factor Use .............................................................. 377Role of Sigma in Promoter Recognition ...................................................... 379

INTRODUCTION

The first step in gene expression is the transcription initiation event, a multistepprocess catalyzed by RNA polymerase holoenzyme. Transcription initiation isa very precise event occurring at specific sites with specific orientations on the

3550066-4197/85/1215-0355502.00

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356 REZNIKOFF ET AL

chromosome. This specificity results from the recognition by RNA polymeraseholoenzyme of DNA sequences termed promoters. The sequence of the pro-moter determines the location and orientation of the 5’ end of the mRNA and isan important element in determining the frequency of transcription initiation.What is the structure of a promoter? One goal of this article is to present ourcurrent answer to this question. We show that although a simple model can beused to define many Escherichia coli promoters, numerous exceptions to thismodel are known. It is thus possible that various alternative promoter structuresexist, possibly reflecting alternative routes in the transcription initiation processfor different promoters, or else we have in general oversimplified our un-derstanding of their structure.

An important means by which gene expression can be controlled is throughthe regulation of transcription initiation. That is, the frequency of transcriptioninitiation as programmed by a promoter sequence can be modulated by a varietyof mechanisms. The most well-known mechanism by which this is accom-plished is through the binding of some other sequence-specific protein nearbyor within the promoter. Other mechanisms of regulating transcription initiationare also known to exist. The second goal of this article is to describe thesevarious regulatory strategies, concentrating on systems found in E. coli. Onemechanism that functions in E. coli, the generation of different specics ofholoenzyme through the use of different ~r subunits, has been extensivelystudied in Bacillus subtilis and is also described. The reader may also beinterested in articles which describe how gene regulation in some systemsoccurs through the control of the transcription termination process (3a, 37a).

PROMOTER STRUCTURE

The primary approach to defining the location and structure of a promoter is theidentification of mutations that alter its recognition by RNA polymerase. Thesemutations alter (either raise or lower) the level of gene expression in cis-dominant fashion and do so by directly affecting the RNA polymerase-promoter interaction.

Genetic analysis of such mutations for bacterial systems provides a simplecommon result. Promoter mutations are located prior to (upstream of) thestructural gene(s) whose expression is being programmed. Classical genetictechniques obviously do not provide the details required to define promoterstructure; instead, one must turn to DNA sequence analysis coupled to thebiochemical definition of the precise mRNA start point. The value of DNAsequence determination is obvious: it provides the ultimate in fine-structuregenetic mapping. The mRNA start-point determination is also critical becauseit provides an orientation point for the promoter sequence; this allows it and itsmutant variants to be compared to other known promoters.


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TRANSCRIPTION INITIATION REGULATION IN BACTERIA 357

-45 +1proposed canonical

promoter sequence 0 cot

lacP mutations

consistent with

canonical model

-35

tcTTGACOt

GACATG T

-I017 bD ~t t tg TAtAaT

AA(+)

+I0

other lacP A(+) T(=) C(+)A(+)mutations

bacteriophage AAAA TTGCT TATAATA CATA TTGATS promoter

homologies

Figure 1 Promoter structure for ~rTM holoenzyme. The top line indicates the proposed canonical

promoter structure for one strand of DNA (57). + I indicates thc position corresponding to the first

base of the mRNA, + numbers indicate downstream sequences, - numbers indicate upstream

sequences. The indicated lacP mutations all decrease lacP expression unless they are followed by a

(+) (increase in expression) or (=) (no change in expression). The mutations are described

References (113) and (120). The bacteriophage T5 promoter homologies were described

Reference (8).

Figure 1 summarizes (57) the general result obtained from this kind genetic, sequence, and mRNA start-point analysis for E. coli promoters recog-nized by ~r7° holoenzyme (Eo"7° is the major E. coli RNA polymerase species;

an alternative species is discussed below). Using the mRNA start point as guide, one can see that there are two sequences whose presence is highlycorrelated with promoter activity. One hexadinucleotide sequence (TATAATin the antisense strand) is centered 10 bp before the start point. A secondhexadinucleotide sequence (TTGACA) is centered approximately 35 bp up-stream of the start point. Although the spacing between the -10 regionsequence and the start point is somewhat variable, there does appear to be adistinctly favored spacing of 17 bp between the -35 and the -10 sequences.The results that lead to the above generalizations are the following:

1. The above commonalities were found in a statistical analysis of more than100 E. coli promoter sequences.

2. Most mutations that alter promoter activity change the particular promoter’ssequence in an expected fashion (see second line of Figure 1). For instance,mutations that enhance the similarity of the particular promoter to theproposed canonical sequence (either by changing the - 10 region, the -35region, or the spacing between the -10 and -35 regions) enhance thepromoter’s activity; additional examples have been recently reviewed (57),


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358 REZNIKOFF ET AL

and the extensive mutational analysis of the phage P22 ant promoter inparticular offers evidence that changes away from the proposed commonsequence decrease promoter activity (151a).

3. In general one can obtain an approximate ranking of promoter activityvis-?~-vis other promoters by comparing their sequences (101).

Obviously other types of RNA polymerases (either completely differentproteins as in the case of bacteriophage-encoded enzymes, or holoenzymescontaining different species of tr subunits) will recognize other sequences aspromoters (examples are discussed below). One expects these sequences to fitinto decipherable general patterns. The surprising observation with the ~r7°

holoenzyme is that there are several exceptions to the proposed generalizedpattern. This, in turn, suggests a caution in developing rigid canonical guide-lines describing promoter sequences for other RNA polymerases; the ex-ceptions may provide us with interesting information about how transcriptioninitiation occurs and how it can be regulated. Some of these exceptionalfindings are described next.

1. Some promoter mutations do not affect any of the canonical promotersequence characteristics described above and in Figure 1. The lac promoterprovides three such examples (see Figure 1, third line); Pqa (at -i6), $7 +6), and prl 11 (at + 10) (25, 91). There are various possible explanations the ways in which these mutations enhance promoter function. H. Bujard’slaboratory performed a comparative sequence analysis using only extremelystrong promoters that program bacteriophage T5 gene expression (8). Thisanalysis extended the canonical sites described above to include an AAAAsequence near -43, a TTGA sequence downstream between + 5 and + 9, and ageneral tendency toward A/T richness (see Figure 1, fourth line). Thus theanalysis that generated the picture presented in Figure I may have missed somesites that contribute to the transcription initiation process, possibly because itincluded too many promoters, weak and strong. Alternatively, other base pairsin the promoter could be recognized by RNA polymerase but might becomesignificant in the overall process only when canonical recognition sites are notpresent.

2. Some promoters are located within DNA sequences that contain otherRNA polymerase binding sites, some of whose sequences overlap with thepromoter in question. The lac promoter is a good example of such an arrange-ment, although it is not unique. As shown in Figure 2, there are two RNApolymerase binding sites that overlap lacP. One site (P2) is displaced 22 upstream from lacP (82, 112, 121). The second site (Pl15), which becomesobvious only as a consequence of a single bp change, is displaced 12-13 bpdownstream from lacP (90, 113). A possible consequence of this type arrangement is that some apparent promoter mutations may act not by changingthe promoter sequence per se but by activating an alternative sequence to


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PII5I PI I

I I p:) -22 I "el I +13

CAP SITE OPERATORFigure 2 Overlapping RNA polymerase binding sites in the lac controlling elements. In additionto the promoter responsible for programming lacP expression (P1), there exist two other sequencesrecognized by RNA polymerase; P2, displaced 22 bp upstream from P1, and Pl15, 13 bpdownstream (82, 90, 112, 113, 121). Mutation Prl 15 results in enhanced CAP-cAMP-independentlac expression, it has no effect on P1 expressiou (A/T--~ T/A (=) change at + 1, see Figure 1), rather activates P115 expression. Many mutations in the -35 region of PI are also in the -10region of P2. Also indicated are the target sites for the CAP-cAMP complex and the repressor.

program transcripts (an example would be the lac mutation Prll5) or bygenerating a competitive RNA polymerase-DNA interaction.

The presence of multiple RNA polymerase binding sites close to a promoteralso suggests the interesting possibility that the alternate sites (or portions ofthese sites) may serve to enhance promoter activity. For instance, a series ofclustered sites that can form loose but specific contacts with RNA polymerase[similar to the product of the first step in RNA polymerase-promoter in-teraction; sometimes called a "closed complex" (80)] might serve as an "an-tenna" to enhance the probability of RNA polymerase molecules being in thelocal environment of the promoter and thus facilitate its productive interactionwith the promoter. This possibility has not been critically examined. Mutationsin such an antenna could enhance or decrease promoter activity.

3. Some promoters appear to lack one of the canonical sequences (a - 10region or a -35 region) without a commensurate effect on promoter activity.An extreme example is the PRE* promoter, a mutant of the kPRE promoter. Itdiffers from the hPRE promoter by three single bp substitutions, which result inthe generation of a "canonical" - 10 sequence. However, PRE* does not appearto have a "-35 region" at all. Moreover, PRE* DNA molecules that end at - 17(totally lacking any DNA sequences corresponding to a -35 region) are able program transcription in vitro. This property is unlike that of any other testedpromoter DNA fragment. It is proposed that in this case a sequence between- 13 and - 17 compensates for the lack of a - 35 region (S. Keitty, Y. S. Ho, G.Sathe, and M. Rosenberg, personal communication).

The lac P115 promoter may be another example. The level of P115 pro-grammed transcription is significantly above that predicted from its sequencerelationship to the canonical sequence (101, 153). The primary discrepancy that P 115 has no appropriately positioned sequence approximating a canonical-35 sequence (101, 113, 153). As discussed below, Pl15 may also have compensating sequence.

4. The canonical promoter sequence model described above predicts thatDNA sequence information upstream of the -35 region should have little or noinfluence on promoter activity. This can be tested by synthesizing or isolating


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360 REZN1KOFF ET AL

deletions that progressively approach the promoter from the upstream region.This type of study with the lacUV5 promoter gives the expected result. Thedeletions have no effect on promoter activity until they actually encroach uponthe -35 sequence (153). However, quite a different result has been found forother promoters. In P115, the approaching deletions have no effect until theyreach position -42, at which point a 3-fold decrease in promoter activity isobserved. These deletions may define an upstream sequence that compensatesfor the lack of a canonical -35 region in P115 (153). A more dramatic resultwas reported for the promoters programming tyrT (71), rrnB (R. Gorse and M.Nomura, personal communication), ilvlH (56), ilvG (C. W. Adams, M. Rosen-berg, and G. W. Hatfield, personal communication) and K. pneumoniae nifLA(28)3 expression. In these cases deletions ending 15 to 145 bp upstream fromthe -35 region decrease promoter expression by up to 10-fold. It is not knownwhether these deletions exert their effect by removing a specific site or by justchanging the overall sequence composition. However, in another case, the hisRpromoter in S. typhimurium, a 3 bp deletion at position -70 (relative to the startpoint) results in a 2-fold reduction in promoter activity (6). Thus in this case specific upstream site is implicated. In both this case and the rrnB promotersituation, the relevant upstream sequences result in an unusual DNA conforma-tion as judged by aberrant electrophoretic mobility of DNA fragments carryingthis region (6; R. Gorse and M. Nomura, personal communication).

How can such long-range upstream effects be explained? We consider threegeneral possibilities. The upstream sequences may not be part of the promoterper se but rather serve a regulatory role. For instance, they may contain abinding site(s) for a positive regulatory protein(s) or a site(s) of action for gyrase such that it generates the proper localized topology for maximal promot-er activity. Alternatively they could represent a part of the promoter. In this casethey might be an extended antenna for RNA polymerase interaction as de-scribed above. Finally these sequences could locally affect nearby DNA con-formation through "telestability" (7a) effects generated by virtue of theirsequence or composition. It is not clear whether this last possibility fits withinthe definition of a promoter or not.

5. Downstream sequences may also affect the activity of some promoters,but except for the point mutations and T5 promoter sequence analyses describedabove, this possibility has not yet been explored.

THE REGULATION OF Eft 7° TRANSCRIPTIONINITIATION

One of the most important conclusions from studies of the last quarter century isthat the regulation of gene activity is often the result of regulating the frequency

3Note: nifLA may be transcribed by an alternate form of holoenzyme as described below.


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of transcription initiation. In the following sections we describe differentmechanisms through which this can be effected. These include (a) the action regulatory proteins that inhibit (repress) transcription initiation; (b) the action regulatory proteins that stimulate (activate) transcription initiation; (c) regulation of DNA topology; and (d) the modification of DNA bases within thepromoter sequence.

Negative Regulation of Transcription Initiation

The first model to be elucidated was that gene expression could be regulated bya repressor protein that, when bound to the DNA, would block transcriptioninitiation. The DNA binding property of the repressor would in turn be mod-ulated by the binding to the repressor of a relevant small molecule effector. Thelactose (lac) operon, shown in Figure 3, was an important system for studyingthis type of regulation, and to a first approximation it suggests an easilyunderstood mechanism that is readily applicable to many other negativelyregulated systems. The three lac genes are expressed at a high level only whenthe cells are grown in medium containing compounds resembling the substratefor the pathway, lactose. Extensive genetic, physiological, and biochemicalexperiments have indicated that this regulation occurs as follows. The lacI geneencodes a repressor protein that when bound to the lac operator inhibitstranscription initiation. This repressor-operator complex is destabilized (andtranscription initiation is allowed) by the binding of any one of a number of13-galactoside compounds (for instance allolactose, an isomer of lactose, orisopropyl-13-t~-thiogalactoside) to the repressor (see 63 and reviews in 5, 95).While many other repressors have their activity modulated by a similar effectorbinding mechanism, some repressors, notably the bacteriophage lambda clrepressor, are inactivated by a proteolytic attack (122a).

How does the repressor act to block transcription initiation? Sequenceanalysis indicates that the repressor binding site (the operator) overlaps with thepromoter such that binding of the repressor and RNA polymerase should be

P T POIOCZ

,/~-galactosidase Lactose ThiogatoctosidePermease Transocetylase

Figure 3 The lactose operon. The transcription of the lacZ, Y, and A genes results fromtranscription initiation by RNA polymerase (RNAP) at lacP. This event is regulated in a positive

fashion by the catabolite gene activator protein (CAP) when it is complexed with cyclic AMP and

a negative fashion by the lacl gene product, the repressor. The repressor in turn is inactivated by theinducer (I). Translation (indicated by the wavy arrows) of each of the product proteins results

ribosome (rbs) binding to the mRNA translation initiation signal for each gene. This figure is the

same as that presented in Reference (120).


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362 REZNIKOFF ET AL

mutually exclusive competitive events (see Figure 2). In vitro transcriptioninitiation experiments by Majors (84) measuring the kinetics of RNApolymerase binding to the lacUV5 promoter suggest that this is precisely themechanism functioning for lac. That is, RNA polymerase does not bind to thepromoter until the repressor is removed from the operator. The location ofoperators in many other negatively regulated systems suggests that a similarmechanism functions there as well.

There are important exceptions to this simple arrangement, two of which areexemplified by the galactose operon system (61, 86):

1. galOc mutations define two gal repressor binding sites, both of which arerequired for maximum efficacy of the gal repressor, and

2. neither of the operators are located in a position that would be presumed toresult in direct competitive binding of the gal repressor and RNApolymerase. One gal repressor binding site is located upstream centered atposition -60; the other is located downstream centered at position +50.The arabinosc BAD operon also shows a dual binding site mode of repressoraction. This includes sites centered at -280 and a site within the araBADcontrolling elements, probably at aral immediately adjacent to the promoter(30).

Surprisingly, the lac system also shows an element of the multiple repressorbinding site pattern, and some of its properties suggest a role for additionalsites. It has long been known that there are three repressor binding sites in lac;the operator, a secondary site with a repressor binding affinity approximatelyten-fold lower than lacO centered at position +413 in the lacZ gene, and atertiary repressor binding site with a still lower repressor affinity centered atposition -82 (41, 122). Deletion of the tertiary binding site has no measurableeffect on lac regulation. Comparable experiments removing the secondarybinding site have not been done, but cloning the lacP-O region away from thesecondary binding site does give constructs that are regulated in a qualitativelycorrect manner. Recent in vitro lac repressor-operator affinity measurementssuggest that the secondary lac repressor binding site may act to stabilize therepressor-operator complex and that this stabilization becomes particularlyapparent in cases where the repressor-operator affinity is weakened by Oc

mutations. That is, the repressor-operator affinity is weaker for Oc mutationswhen the secondary binding site is missing than when it is present (100). Thissuggests that secondary repressor binding sites in general may act by stabilizingrepressor-operator complexes and that these secondary binding sites do notdirectly participate in the inhibition of transcription initiation.

We are still left with the second question, how does the gal repressor functionsince in this case the presumed primary repressor binding site does not overlapwith the promoter sequences. It is possible that when bound to both binding


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sites simultaneously, the repressor holds the DNA in a conformation that isunfavorable for productive binding of RNA polymerase.

Positive Regulation of Transcription InitiationStarting with the studies on the arabinose (ara) regulon by Englesberg and hiscoworkers (see 73 for review), it was discovered that several systems are

positively regulated. That is, the frequency of transcription initiation is en-hanced by an activator protein. The activation of lacP expression by the

catabolite gene activator protein-adenosine 3’ 5’-cyclic monophosphate(CAP-cAMP) complex is the model system we use to illustrate this type regulation (see Figure 3). In addition to the lac operon-specific control ex-emplified by the repressor-operator system described above, the maximal level

of lac expression (and that of several other operons encoding catabolic func-tions) is modulated by the presence of (or, to be more precise, transport of)other available carbon sources in the media. Three classes of mutations havebeen isolated that prevent maximal level of lac expression and that definemolecular components participating in this type of regulation.

1. Mutations in the crp gene that fail to make a functional CAP. This protein

is known to be an activator for the expression of lac and other systemscontrolled by catabolite repression (24, 33, & 110).

2. Mutations in the cya gene that fail to synthesize adenyl cyclase (127). Thecya mutants do not make cAMP. In vitro experiments have shown that cAMP isrequired for activation of CAP (24, 33).

3. Cis-dominant mutations associated with each regulated system that re-duce the maximal stimulatory response of CAP-cAMP (4, 26, 152). Some these cis-dominant mutations define the DNA target site for CAP. This site hasalso been defined by biochemical experiments (126, 130, 185; also see Figure2). As discussed below, the biochemical definition of this site is critical. Thisdefinition includes CAP-cAMP-IacP DNA binding experiments that tested theeffect of CAP site mutations on this reaction, and experiments that usedchemical probe and nuclease protection protocols to examine the precise DNAbinding site for CAP (85, 126, 130).

-.30 t -,20 t -.10 t ÷.1t -.20ti -.lOti *,lti

AACGGAACCTTTCCCGTTTTCCAGGATCTGATCTTCCATGTGACCTCCTAACATGGTAACGTTCATGATAACTTCT

a~ --’~ [ ti mRNAt mRNA t protein

Figure 4 Tn5 ~ransposase and transpos~lon ~nh~b~tor promoters. The ISS0 inse~ion sequencebracketlng Tn5 contains two promoters tha~ program ~he synthesis of the two protelns lnvo]ved in~ran~po~l~lon. The promoter for ~he ~ransposase (~) ls expressed a~ low levels unless ~he indlca~eddo~ sltes are unmethylated (L C.-P. Yin, M. Krebs and W. S. Reznikoff, unpubl~shod results).The relevant slgna]s for the transposition ~nhlb~tor promoter are lnd~cated by


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364 REZNIKOFF ET AL

Three important observations arise from studies on the CAP-cAMP DNAtarget site.

1. CAP-cAMP DNA target sites in a number of systems can be defined, andcomparative sequence analyses suggest that these sites approximate the se-quence AA-TGTGA-~-CACA-T (23, 30a).

2. CAP-cAMP DNA target sites are found at different locations vis-h-vis thetranscription start site in different systems (23).

3. In lac, other mutations have been isolated with the same Lac phenotype(Lac- because lac expression does not respond properly to CAP-cAMP) but arenot in the biochemically defined CAP-cAMP target site. These are "spacer"mutations; insertions or deletions that change the distance between the CAP-cAMP target site and the promoter and change the structure of the overlappingupstream RNA polymerase binding site P2 (89, 152),

Unlike the situation with negative regulation, a mechanism for positiveregulation is not intuitively obvious. (Of course, our intuition about negativeregulatory mechanisms may be misleading for some systems). Below wediscuss three mechanisms by which activator proteins might act.

1. An activator protein might act by perturbing the conformation of the DNAextending into the promoter (25). This "telestability"-like effect could, forinstance, lower the energy requirements for open complex formation. [Thepossible steps in the transcription initiation process have been discussed else-where (80)]. There is no evidence specifically supporting this model. It compatible with the fact that CAP-cAMP target sites are located differently indifferent systems. In this case the spacer mutations might alter a sequencenecessary for the propagation of the "telestability" effect.

2. The activator protein might act as a contact point for RNA polymeraseand thereby facilitate its formation of a productive open complex (40). Thereexists compelling genetic evidence that this is the mechanism by which the h cIprotein activates expression from promoter PRra (51). A missense mutation hasbeen isolated in the cI gene that results in the synthesis of a product with normalDNA binding properties but that fails to stimulate transcription initiation fromPRr~. A cI protein model generated from X-ray crystallographic data suggeststhat the altered residue is appropriately positioned for forming a contact withRNA polymerase bound to PRr~ (60).

The relationship of this model to CAP-cAMP’s mode of action is less clear.The fact that CAP-cAMP target sites are located in different positions vis-a-visthe promoter in different systems suggests that CAP-cAMP does not stimulatetranscription by means of interaction between CAP-cAMP and RNApolymerase. However, this model is not ruled out because of the followingconsiderations. In some systems, such as gal, the CAP-cAMP target siteappears to be appropriately positioned in order to facilitate the proposedprotein-protein contact. In other systems, such as the araBAD operon, the


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regulatory sites in the DNA have the following arrangement. The CAP targetsite is next to the target site of the ara activator protein (araC), which is in turnnext to the promoter. Such an arrangement might facilitate a protein (CAP-cAMP)-protein (araC)-protein (RNA polymerase) interaction. Left unresolvedare situations such as lac in which the CAP-cAMP target site is apparentlylocated too far from the promoter to interact directly with an RNA polymerasebound to lacP, and yet there is no known intervening binding site for anotherregulatory protein. One way to resolve this discrepancy would be to proposethat CAP-cAMP interacts with RNA polymerase, which binds to an upstreamoverlapping RNA polymerase binding site, P2, and facilitates the movement ofRNA polymerase from P2 to the promoter (89; see Figure 2). In this light it interesting to recall the "spacer" mutations in lac, which reduce the efficacy oflac stimulation by CAP-cAMP. Those spacer mutations which have been testedreduce the affinity of RNA polymerase for P2 (154).

3. The activator protein might act as a repressor of a protein-DNA bindingreaction that would otherwise inhibit the RNA polymerase-promoter interac-tion. This was initially proposed to partially explain how CAP-cAMP mightactivate lac (82, 87, 121). If one assumes that P2-bound RNA polymeraseinhibits RNA polymerase interaction with lacP, then CAP-cAMP might beenvisioned as blocking this inhibition. The properties of the "spacer" mutationsin lac suggest that, for this specific case, this model is not correct. The spacermutations reduce the RNA polymerase-P2 affinity. If the competitive modelwere correct, one would predict that these mutations would enhance CAP-cAMP-independent lac expression. Such an enhancement is not observed(154). There is however, another system in which such a cascade of competitivebinding events may occur. This involves the CAP-cAMP stimulation of hutUHin K. pneumoniae. The hutUH controlling elements contain two divergentpromoters that overlap and appear to compete with each other for RNApolymerase binding. The CAP-cAMP target site overlaps with the upstream-facing promoter. Binding of CAP-cAMP to this site appears to inhibit RNApolymerase binding to the upstream-facing promoter and stimulate its binding

- to the downstream-facing promoter (107).

DNA Topology Regulation of Transcription Initiation

The in vivo template for transcription is known to be a negative superhelix.Since the presence and amount of superhelicity affect the energy required todenature any region in the molecule, and since RNA polymerase-promoteropen complex formation involves localized DNA melting (128), changing thesuperhelical character of the template would be expected to change its promoterproperties; therefore, this might be a mechanism used for regulating transcrip-tion initiation. In fact, as reviewed by Gellert (39), the superhelical character the template does affect its template properties (although sometimes in


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366 REZNIKOFF ET AL

surprising fashion; many promoters are stimulated by negative superhelicity butsome are inhibited). Effects of superhelicity on the process of transcriptioninitiation have been shown in vitro in numerous studies [for instance, seeBotchan (6a) and Malan et al (87)] and, through the use of gyrase inhibitors, vivo by Sanzey (125). There is one case in which the regulation of transcriptioninitiation in response to variations in superhelical density is believed to bephysiologically important. The expression of the gyrA and gyrB genes encodingthe subunits of gyrase (whose function it is to generate supercoils) is enhancedby inhibiting gyrase activity (93). This observation suggests that gene regula-tion may play an importa,nt role in the homeostatic control of chromosomesupercoiling.

DNA Modification and the Regulation of Gene Expression

A frequently invoked model for gene regulation in eukaryotes involves thepresence or absence of methyl groups on the DNA. Some bacterial systemsclearly use this mechanism (131a).

The effect of DNA methylation on promoter activity was first suggested byexperiments of Denise Roberts and Nancy Kleckner in studies of transpositionof Tnl0 (personal communication and 67). Host mutants were isolated thatenhanced the frequency of Tn 10 transposition. Some of these were found to bein the dam gene, which encodes the methylase that forms N6-methyl adenine atthe DNA sequence GATC. The enhanced Tnl0 transposition in these mutantswas found to be correlated with the enhanced synthesis of the transposasemRNA. The same basic mechanism appears to be functioning in Tn5 (J. C.-P.Yin and W. S. Reznikoff, unpublished results). As shown in Figure 4, thesequence of the Tn5 transposase promoter suggests a reason for these observa-tions. In these transposons the transposase promoter contains either one (Tn 10),or two (Tn5) dam sites within the promoter. It is proposed that methylation ofthese sites inhibits the recognition of these promoters by RNA polymerase. Asimilar presence of dam sites has also been observed for some promoterslocated in the bacteriophage P1 genome (13 l a).

If one assumes that hemimethylated DNA also has altered protein-DNArecognition properties in these cases, this dam site modification mechanismprovides an intriguing means by which gene expression could respond to (a) thepassage of the replicating fork, (b) the repair of damaged DNA, and (c) transfer of DNA via conjugation. If no methylation of both strands is required,this system would also have interesting implications for its int~’oduction intoheterologous hosts; not all bacteria have a darn system (3).

MODIFICATION OF HOLOENZYME STRUCTURE ANDTHE REGULATION OF GENE EXPRESSION

The first evidence for dissociable RNA polymerase subunits required forselective initiation of transcription came from the discovery of ~r7° by Burgess


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and Travers (10, 11). E. coli RNA polymerase, purified to homogeneity bychromatography on a phosphocellulose column (9), is highly competent transcribe calf thymus DNA, but is unable to transcribe T4 DNA efficiently. Asearch for the factor that permitted such transcription resulted in the discoveryof the ~r7° subunit (10, 11), and the recognition that RNA polymerase exists two forms: holoenzyme (Eo"7°) capable of selective initiation at promoterregions, and corc RNA polymerase (E) capable of elongation and terminationbut not of selective initiation. This characteristic of RNA polymerase, that theability to initiate transcription selectively is dependent on the presence of the ~rsubunit, suggests that the specificity of gene expression could be modulated bysubstituting one species of ~r for another (10). Below we show that this is indeedthe case and that this is an important means of regulating gene expression.

The discovery of ~7o was made possible by two things: (a) the existence of template containing promoters whose transcription required that sigma factor,and (b) the existence of a procedure for separating the sigma subunit from coreRNA polymerase. Since that time, other sigma factors, encoded either bybacterial or phage genomes, have been purified. In each case their identificationdepended on the ability to separate the putative sigma subunit from core RNApolymerase and the existence of a template capable of demonstrating thespecific promoter recognition properties of the reconstituted holoenzyme.

The sigma factors are used to regulate genes with diverse biological roles.The first alternative sigma factors were identified in B. subtilis and its phages(29, 37, 138, 139) and were implicated in the temporal regulation of develop-ment (reviewed by Losick & Youngman, 79). More recently, alternative sigmafactors were also implicated in cellular responses to various environmentalstimuli. Below, we briefly describe the biological role of each sigma. Weconclude by considering the interactions of the sigma factors with each other,the similarities among sigmas, and the role of sigma in selective initiation.

E. coli (~70

The tr 7° subunit of RNA polymerase is known to be essential for cell growthsince mutations in rpoD (encoding ~r7°) confer a ts growth phenotype (55, 75,102, 109). ~r7° is required for most transcription; cells containing the tempera-ture-sensitive sigma mutation, rpoDSO0, cease expressing most cellular genesshortly after temperature upshift (48).

E. coli cells contain about 3000 molecules of ~7o, enough to bind about onethird of the intracellular core RNA polymerase (32, 62). On the basis comparisons of in vivo and in vitro transcription rates, McClure (81) estimatedthat only about 1% of the RNA polymerase holocnzyme is present as freepoly~nerase. The remaining holoenzyme is sequestered by nonspecific DNAbinding and acts to buffer the in vivo concentration of free polymerase. Thisnotion is consistent with the observation that, although gene expression islimited by transcription initiation, increasing the absolute concentration of ~r7°


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368 REZNIKOFF ET AL

increases neither the overall rate of transcription nor the synthesis of majorcellular proteins (103).

The Heat Shock Response and cr~2

The heat shock response in E. coli is regulated by the product of the htpR (rpoH)gene (105, 151), which was recently shown to be a sigma factor, 32 (50).When cells are shifted from low to high temperature, the synthesis of a smallnumber of proteins, the heat shock proteins, transiently increases. The heatshock response in bacteria was recently reviewed (49, 106).

The criteria that demonstrated the rpoH(htpR) gene product to be a sigmafactor were similar to those described above (50). The factor responsible fortranscription of a heat shock gene promoter was purified from crude extractsand separated from core RNA polymerase by electrophoresis on SDS-polyacrylamide gels. Upon renaturation and reconstitution with core RNApolymerase, the new form of holoenzyme, E~r32, was shown to initiatetranscription from heat shock gene promoters, but not from two strong Ecr7°

promoters: PlacUV5 and the RNA I promoter of ColE1 type plasmids (49, 50).A comparison of the amino acid sequences of ~r32 and crv° shows that they arevery similar, consistent with the identification of cr32 as a sigma factor (72,155). (The regions of homology among sigma factors are discussed below).

Five heat shock gene promoters, which precede the heat shock genes rpoD,dnaK, groE and the C62.5 gene, have been characterized (20, 137). Transcrip-tion initiates from these promoters both at low temperature and after a shift tohigh temperature, but the amount of transcription increases after the shift tohigh temperature. These promoters are recognized in vitro by holoenzymecontaining ~r32, Ecr32, but not by Ecr7° (20; Table 1). These promoters share consensus seque,nce having T-tC-CcCTTGAA in the -35 region andCCCCATtTa in the -10 region.

The mechanism regulating the increase in transcription initiation by Eo"32

after heat shock is unknown and is currently under study in several laboratories.Two alternative classes of models can explain enhanced initiation at heat shockgene promoters. In the first class, activators or repressors acting at the heatshock gene promoters and affecting the frequency of initiation by Ecr32 arealtered by the inducing stimulus. There is no evidence for auxiliary factors;however, an exhaustive search has not yet been carried out. In the second classof models, either the amount or activity of ~r32 itself transiently increasesrelative to other sigma factors in the cell. In this case, the amount of ~r32 mustlimit the transcription of even the strongest of the heat shock promoters. Thishas been shown to be true. cr32 is normally present in very small amounts (F. C.Neidhardt, personal communication; A. Grossman, unpublished data). When~r32 is overproduced from a foreign promoter, the rate of synthesis of the heatshock proteins increases without a temperature upshift (A. Grossman, manu-script in preparation).


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370 REZNIKOFF ET AL

0-32 is very unstable in vivo, with a T~/2 of about 3’ (A. Grossman, personalcommunication). Thus, changes in the rate of synthesis of 0-32 can rapidly alterits intracellular level. While these findings are consistent with the second classof models, they do not prove such a model.

The E. coli ntrA(glnF) Protein: Another Sigma Factor

When enteric bacteria (for example, E. coli, Salmonella, and Klebsiella) arenitrogen limited, the synthesis of a number of proteins (including glutaminesynthetase and amino acid transport components and degradative enzymes) isinduced 10-100-fold. The increased production of these proteins enlarges thecapacity of cells to produce glutamate, the major precursor of other nitro-gen-containing compounds. Some organisms (for example, Klebsiella) also in-duce nitrogenase and other proteins required for nitrogen fixation (83, 94, 143).

Genetic studies established that normal expression of the nitrogen-regulatedgenes requires both the ntrA(glnF) and ntrC(glnG) gene products, whichfunction in vivo as positive regulators. In the absence of these gene products,expression of nitrogen-regulated genes is very low and does not respond to theimposition of nitrogen limitation (70, 83, 94). Because cells lacking NtrCexpressed glnA (encoding glutamine synthetase) at a higher rate than thoselacking NtrA, it had been argued that NtrC also functioned as a repressor.Analysis of the in vivo transcription rates of the glnA gene using Mud-1 fusionsestablished that both NtrA and NtrC work at the transcription level (68, 83,123). Finally, analysis of the 5’ ends of in vivo RNA provided a basis for molecular understanding of the dual action of NtrC (27, 119). These studiesshowed that glnA is transcribed from two promoters (27, 119). In addition,there is a promoter internal to the glnA operon (69, 83, 118,144). Comparisonof transcription from ntr÷ and ntr cells (118, 144) established (a) that represses transcription from the weak, upstream promoter and the internalpromoters but i’s required for activity of the strong downstream promoterresponsive to nitrogen regulation; and (b) that NtrA is required for activity the downstream promoter but does not affect transcription from the upstreampromoter.

The probable biochemical basis of NtrC and NtrA action has been elucidatedby in vitro studies utilizing glnA as a template. It appears that NtrC is a DNAbinding activator protein analogous to CAP (1, 59, 118) and that NtrA probably a new sigma factor (59). Footprinting experiments of Kustu andcolleagues (59, 149) showed that NtrC binds with differential affinity to fivesites upstream of the glnA gene. One of the tightest binding sites overlaps theweak upstream promoter and may account for the ability of NtrC to repress itstranscription (59, 118). The other sites are upstream of the strong regulatedpromoter. Binding at these sites may activate the promoter. Using both anS30-based transcription-translation system and a transcription system, Kustu


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and collaborators showed that partially purified NtrA promotes transcriptionfrom the downstream regulated promoter upon addition of core RNApolymerase and NtrC. Since ¢r7° is not present in the fraction and is not requiredfor this reaction, this fraction must contain a new sigma factor (59). Consistentwith this idea, the sequence upstream of the 5’ end of the RNA (see Table 1)bears no resemblance to an Eo"7° promoter but shows extensive homology toseveral other ntrA dependent promoters, including eight involved in nitrogenfixation (27, 59, 119). It is apparently the ntrA product itself which stimulatesglnA transcription rather than another protein whose synthesis depends on thentr system: Addition of an ntrA÷ plasmid to an ntrA- $30 resulted in a 30-foldstimulation of glnA expression and since protein synthesis in $30 extracts isdependent on an exogenous DNA template, stimulation was presumably due tontrA product synthesized from the plasmid (J. Keener and S. Kustu, un-published).

How nitrogen limitation results in enhanced expression of nitrogen-regulatedgenes is not known. Transcription of ntrA does not increase under nitrogen-limiting conditions, which suggests that the amount of NtrA does not regulatethe response (22). On the other hand, the amount of NtrC does increase whencells are limited for nitrogen (69, 83, 118). In addition, some mutationsmapping in the ntrC gene result in high-level constitutive production of nitro-gen-regulated proteins (70). Taken together, these observations suggest thatincreases in the amount or activity of NtrC could result in the increasedtranscription of nitrogen-regulated genes. If so, the sensor of nitrogen limita-tion may well affect the NtrC gene product.

B. subtilis Sporulation

It had long been known that RNA polymerase isolated from sporulating Bacil-lus subtilis cells had a different polypeptide composition than RNA polymerasepurified from vegetative cells. This led to the speculation that new sigmas orother transcriptional factors might be responsible for altered transcriptionalpatterns during sporulation (78). We present a brief description of the sporula-tion process in B. subtilis. We then describe how each sigma in B. subtilis wasidentified and the current ideas about the biological role for each sigma.

The precise stimuli inducing spornlation are unknown; however, it is effi-ciently induced by nutrient deprivation. This developmental process has beendivided into several phases based upon morphological landmarks. One of theearliest steps is invagination of the plasma membrane that divides the sporulat-ing cell into a mother cell and a forespore compartment (35). The fore’spore engulfed by the mother cell membrane, develops a cortex and tough proteincoat, and is eventually released as a dormant spore when the mother cell lyses.Both the spore and the mother cell contain a transcriptionally active chromo-some (124), and there is some suggestion that gene expression is com-


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372 REZNIKOFF ET AL

partmentalized (74, 104). Mutations are known that arrest cells at variousstages of sporulation. Genes identified in such a manner are named for the arreststage; for example, spolIA mutants arrest at the second stage. Mutations thatarrest the sporulation process before visible landmarks have occurred aretermed spoO mutants. Although some of the spoO mutants have altered pheno-types during vegetative growth, none of the spoO gene products has been shownto be essential for vegetative growth.

Sigma Factors in B. subtilis

The most abundant sigma factor in B. subtilis during vegetative growth is 0"43,

encoded by the rpoD gene located at 225° on the B. subtilis map. The gene wasrecently cloned and sequenced by Gitt et al (45). The change in nomenclature

from 0.55 to 0.43 reflects the corrected M.W. assignment based upon DNAsequence determination. Sequence comparisons have established that 0.43 and0.70 are homologous (45; also, see below).

It is presumed that the role of E0.43 is to transcribe most of the genesexpressed during the vegetative state, but there are no conditional lethalmutations in the B. subtilis rpoD gene to test this assumption. The only existingrpoD mutations are the crsA mutations (133a; R. Doi et al, personal com-munication). The presence of glucose usually inhibits sporulation. Cells withcrsA mutations sporulate even when glucose is present in the medium. Themolecular mechanism of this effect is not understood.

The consensus sequence for promoters recognized by Eo"43 iS identical to thatderived for E. coli Eo"7° (96, 98; Figure 1 and Table 1). However, strongpromoters in B. subtilis appear to require additional information. For example,the tac promoter, a very strong promoter for E. coli E0.7° that has the consensussequence at both the - 10 and -35 regions of the promoter with a spacing of 16bp, is not recognized in vitro by B. subtilis E0.43 (96, 98). Possibly, B. subtilisE0.43 has very rigid spacing requirements. To date, all Eo"43 promoters identi-fied have a spacing of 17 or 18 bp. Two additional features of strongB, subtilispromoters have been noted: an extremely AT-rich region upstream of thepromoter and the sequence RTRTG at positions -14 to -18 (96, 98). Theimportance of these features for recognition by Eo"43 has not been tested.However, the AT-rich region upstream of several developmentally regulatedgenes has been found to enhance expression without altering regulation (2; R.Losick, personal communication), suggesting that the AT stretch may be general structural feature of strong B. subtilis promoters.

0.28 is a minor sigma factor present in vegetatively growing B. subtilis. The

discovery of 0.28 is a prototypic example of how sigma factors are definedbiochemically. Chamberlin and his collaborators (64) found that B. subtilis butnot E. coli RNA polymerase makes a unique transcript from T7 DNA, termedthe J transcript. Using the ability to make this transcript as an assay, they


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purified the enzymatic form with unique promoter recognition properties (64,148). It proved to be a novel form of holoenzyme containing a 28-kd proteinrather than the normal 43-kd sigma factor protein. The 28-kd protein wasdissociated from E~28 and shown to be responsible for the novel promoterrecognition properties when reconstituted with purified core, which thus rigor-ously established that the protein was a sigma factor. The gene encoding thisprotein has not been identified. The number of strong Err28 promoters has beenestimated at 20-30 (43).

To dissect the biological role of ~r28, Gilman & Chamberlin (42) made use the fact that this form of holoenzyme has a stringent promoter preference andhas virtually no activity on templates lacking cognate promoters. They identi-fied two B. subtilis DNA sequences recognized as promoters by E~r28 in vitro(44) and showed that these promoters were also utilized in vivo. Gilman Chamberlin (42) showed that these promoters were transcribed at a very lowrate during vegetative growth and regulated by the sporulation machinery. Thetranscripts are absent in four different spoO mutants, spoOA, B, E, and F, andare shut off immediately when exponential growth ends (preceding shutoff ofErr55 transcripts by one hour). Note that if all other tr28 transcripts are similarlyaffected by the spoO loci, tr 28 must not be essential for vegetative growth.

To determine if the lack of the tr 28 transcripts following the end of ex-ponential growth results from the loss ofE~r28, Wiggs et al (147) compared theamount of E~r28 in growing and post-exponential cells and the amount of E~r28

in spoOA mutant and wild-type cells by following Err28 activity after a one-steppolymerase purification. They found reduced amounts of E~r28 activity inpost-exponential cells. In the absence of an immunological assay, it is difficultto know whether this reduced activity reflects lack of tr 28 in the cell or theinability of ~r28 to associate with core RNA polymerase. The latter possibilityhas been shown in the case of ~r43, which is present during sporulation but is notfound in polymerase prepared from sporulating cells (139). In contrast, Wiggset al (148) recovered equivalent amounts of E~r28 from wild-type and spoOAmutants. These results suggest that the spoOA locus (and possibly other spo locias well) encodes a protein required for E~r28 activity in vivo. An alternativepossibility, that one of the spo loci encodes yet another sigma factor thatactually reads these promoters in vivo, has not been excluded.

Briat et al (7) recently showed that E~r~8 recognized the E. coli heat shockpromoter rpoD Phs in vitro, which indicates that E. coli Eo"32 and B. subtilisE~r28 may have overlapping promoter specificity. It is also possible that thesetwo ~r factors have homologous roles. Preliminary studies indicated that the twoidentified tr 28 promoters did not heat shock in either E. coli (7) or B. subtilis(42). Gilman et al (43) recently isolated a new set of strong 28 promotersfrom B. subtilis. It will be interesting to see the response of these promoters to atemperature shift.


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374 REZNIKOFF ET AL

Holoenzyme containing 0.37 is present during vegetative growth and earlyduring sporulation. Starting with a template containing spoVG, a gene that isactively transcribed early during sporulation and whose expression is undercontrol of several spoO genes, Haldenwang & Losick (53, 54) purified theactivity responsible for transcribing spoVG from extracts of cells early insporulation (T1). They showed that RNA polymerase purified from theseextracts contained a novel 37-kd protein. This protein was separated from otherRNA polymerase subunits by chromatography on phosphocellulose followingdenaturation in urea. When it was renatured and reconstituted with core RNApolymerase, the reconstituted enzyme was able to initiate transcription from thespoVG promoter; this established that this 37-kd protein was a sigma factor. Invitro E0.37 also transcribes ctc (54), encoding a protein of unknown function,and sprE, encoding subtilisin (150). In vivo, all three genes are transcribedmore actively during stationary phase than exponential growth (99, 108,146).Transcription of spoVG and sprE, but not ctc, is under control of several spoOgenes (97, 108, 114).

The promoter sequences important for E0.37 recognition have been character-ized by Moran and coworkers. A consensus sequence for E0.37 promoters wasproposed based on homology between the ctc and spoVG promoter regions(99). To confirm the importance of this sequence and further define its features,Moran and colleagues analyzed the interaction of Eo"37 with the ctc promoter byfootprinting, DMS protection experiments, and deletion and bisulfitemutagenesis of the cloned promoter. They find that the holoenzyme bindsimmediately upstream of the 5’ end of the gene and protects a region of DNAthat includes the consensus sequence (96). The G residues on the nontrans-cribed strand at positions - 14, - 15, and - 16 (within or adjoining the - 10consensus sequence) and at position -36 (within the -35 consensus sequence)were protected from methylation when Eo"37 was bound to the promoter region(96). Alteration of these same GC base pairs to AT base pairs by bisulfitemutagenesis weakened promoter activity both in vitro and in vivo (117, 135,136).

The biological role of 0-37 has not yet been determined. Although the threegenes identified as 0.37 templates in vitro are not transcribed by E0.43, each isalso transcribed by one or more alternate forms of holoenzy~ne, sometimes withidentical start points (spoVG is also transcribed by E0.32; ctc by both E0.32 andE0.eg) (65,136,146). The in vitro studies make it unlikely that E0.43 transcribesthese promoters in vivo, but they do not establish which alternate form(s)transcribe these promoters in vivo. Identification of the gene encoding 0.37 andisolation of mutants will help to elucidate the role of 0.37 in B. subtilis develop-ment.

~32 is a very-low-abundance sigma factor initially identified as a contaminat-

ing activity in an E0.37 preparation (58, 65). While relatively impure E0.37


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transcribed the spoVG gene from two start points separated by 10 bp, a morepurified preparation of E0.37 used only the upstream start. This difference wastraced to a minor band of 32 kd, visible only upon silver staining the gel. Whenthe 32-kd protein was eluted from the gel, renatured, and reconstituted withcore RNA polymerase, the resultant holoenzyme specifically transcribed thespoVG gene from the downstream start. The biological role of this form ofholoenzyme has not been investigated further.

Sporulating cells contain a 29-kd sporulation-specific protein associated withRNA polymerase (76, 104). Haldenwang et al. (52) purified RNA polymerasecontaining the 29-kd protein and used it to transcribe cloned DNA containingseveral sporulation genes to see if they could detect a novel transcript. Both thectc and spoVG genes described above were transcribed by this holoenzymeform. In addition, a unique transcript termed L was obtained. They separatedthe 29-kd protein from the other RNA polymerase subunits by SDS gel elec-trophoresis, renatured it, and reconstituted it with core RNA polymerase. Thereconstituted enzyme showed the same transcription properties as RNApolymerase containing the 29-kd protein; hence this protein is a sigma factor.

Haldenwang and collaborators have monitored the accumulation of 0-29

throughout the B. subtilis life cycle by using a monoclonal antibody specific to0.29 (140--142). .29 is not detectably present in vegetative cells and is present insignificant amounts in sporulating cells only between stages T2 and T4.Accumulation of 0-29 is under sporulation-specific control and requires thewild-type gene products encoded by the spoO A, B, E, F, and H loci. Themonoclonal antibody to 0.29 detected an additional protein of 31 kd in B. subtilisextracts. Like 0"29, P31 was present only in sporulating cells. The accumulationof P31 preceded that of 0-29 by one hour and the two proteins had related peptidemaps, which suggests a precursor-product relationship for the two proteins.

It has now been shown that 0.29 is encoded by the spoIIG gene (133, 140-142)and is almost certainly synthesized as a 31-kd precursor molecule. Stragier et al(133) showed that the spolIG gene encodes a protein of about 27 kd,homologous to the 0.70 protein of E. coll. Based on the M.W. of the openreading frame, they suggested that the locus might encode 0-29. Two spoIIGmutants were shown to lack both 0-29 and P31. Significantly, one of the mutantsproduced immunologically reactive fragments of 25 kd and 21 kd, whichsuggests that this spoIIG mutation causes premature translation termination.When the spollG plasmid was put into this mutant strain, proteins of 31 kd and29 kd were detected, as well as the endogenous 25 kd and 21 kd proteins. Whenthe spolIG plasmid was put into E. coli, only the 31-kd protein was detected.These observations are most easily explained by assuming that the 31-kdprotein is the gene product of the spollG locus, which is processed to a 29-kdprotein in B. subtilis.

0.29 is the first sigma factor with a known role in sporulation. It is a


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stage-specific sporulation protein encoded by the spolIG locus and is syn-thesized in a precursor form. Mutations in spollG result in arrest at the secondstage of sporulation; the cell is divided into the forespore compartment andmother cell by the septum, but a detached spore within the mother cell does notform. It has been reported that E0-29 is found only in the forespore compartment(104). -29 may be processed concomitantly with t ransport from mother cell t oforespore. Alternatively, the processing machinery may be active only in theforespore compartment. Since P31 associates only weakly with RNApolymerase and does not confer the ability to transcribe E0-29 promoters, the cell

lacks 0-29 until the precursor is cleaved (141). The processing of P31 to 0-29 maybe intimately connected with the regulation of sporulation. One spo locus hasbeen implicated in the processing event; spollC mutations accumulated P31 butnot 0-29,

Since ~rz9 is present for only a short time during sporulation, it is likely thatother sigma factors will be implicated in the sporulation cascade. A potentialsporulation sigma factor is the spollAC gene product which is 22 kd andhomologous to E. coli cr7° (36). It is not known which sigma factor is theproduct of the spollAC gene.

Phage-Encoded Sigma Factors

The first evidence that gene expression is regulated through the use of alternatesigma factors came from work on bacteriophage. Both E. coli and B. subtilisphages use alternate sigma factors for temporal regulation of phage geneexpression.

B. subtilis phages SP01 and SP82 and coliphage T4 all encode three classesof transcripts: early, middle, and late (reviewed in 38, 111, 115). In all casesearly transcripts are made by host RNA polymerase. Production of middle andlate transcripts requires phage-specified proteins.

Transcription of SP01 middle genes requires the expression of phage gene28; appearance of late transcripts is dependent on gene products 33 and 34. Theproducts of genes 28, 33, and 34 have been isolated and shown to function assigma factors (19, 29, 138). When gp28 overproduced in E. coli is reconstitutedwith either B. subtilis or E. coil core, the holoenzyme formed transcribes twomiddle promoters (19). Production of SP01 late RNA in vitro using total SP01DNA as a template, requires the addition of gp33 and 34, as well as the deltaprotein, to core enzyme (138).

The related B. subtilis phage SP82 encodes proteins analogous in size andfunction to gp28, 33, and 34 of SP01. These proteins are found associated withRNA polymerase after infection by SP82 (131) and are likely to also be sigmafactors. Achberger and Whiteley (0) showed that the 28 kd protein from SP82functions as a sigma factor and allows both B. subtilis and E. coli core RNApolymerase to recognize SP82 middle promoters.

RNA polymerase purified 5-10 minutes after T4 infection contains five


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phage-encoded proteins (115). Two of these proteins are the products of genes 33 and 55. The product of gene 55 has been shown to be a sigma factor(66, 88). In vivo transcription from T4 late prrmoters requires both gp33 andgp55, but in vitro, purified gp55 and core RNA polymerase isolated fromuninfected E. coli is sufficient for transcription from the late promoter P23 (66).The function of gp33 has not yet been determined.

~b29 is a lytic phage of B. subtilis that encodes two temporal classes ofmRNA. Appearance of late mRNA requires the product of d¢29 gene 4 (re-viewed in 38). Mellado et al. (92) have overproduced gp4 E. coli. Additionof the partially purified protein to B. subtilis core RNA polymerase is requiredfor synthesis of two transcripts from the late region of the ~b29 genome. Thesetranscripts are not made when 429 DNA is transcribed by either corepolymerase alone or core supplemented with extracts containing a mutant gp4,suggesting that gp4 is a sigma factor.

Conservation of Structure Among Sigma Factors

In the past several years, the sequence of nine sigma factors has been reported:0.70 (12) and 0.32 (72, 155) from E. coli; 0.43 (45), .29 (133), and spollAC (36)

from B. subtilis; 0.gp55 (46) from E. coli phage T4; ~rgp28 (18) and ITgp34 (17)from B. subtilis phage SP01; and the p4 protein from B. subtilis phage 29 (34).The derived amino acid sequences have been compared using a variety ofcomputer programs designed to identify evolutionary relationships betweenproteins.

The two E. coli sigma factors were the first to be identified as beinghomologous. Landick et al (72) and Yura et al (155) found substantial similaritybetween 0 .32 and the C-terminus of 0.70. Overall, the two proteins share(including identical amino acids and conserved replacement) 44% of theiramino acid residues in this region. One region of 14 amino acids is completelyconserved between the two proteins. In addition, 0.3~ was found to have tworeasonably good matches to the helix-turn-helix motif characteristic of DNAbinding proteins. Subsequently, 0.43 from B. subtilis was sequenced and com-pared to these two proteins. In addition to similarity in the C-terminus, 0.43 alsoexhibited similarity to 0.70 in the N-terminus (45). .29 (133) and the spolIACgene product (47) also exhibit substantial similarity to the C-terminus of 0.70.More recently, eight of the sigma proteins have been compared by Gribskov &Burgess (47). Their results, presented diagramatically in Figure 5, show that alleight of the proteins are homologous. All the proteins except T4 gp55 have atleast one region strongly resembling the helix-turn-helix motif (regions 3 and in Figure 5).

Modulation of Sigma Factor Use

Changes in sigma factors have been implicated in temporal regulation duringirreversible processes such as sporulation and phage development and transient


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378 REZNIKOFF ET AL

0 ~00 200

Figure 5

2

2

5 4Sicjma - 70

5 4SItjma - 45

Sigma- :52

2 4Sigma - 29

2 5Spo]I Ac

2 4SPOI qp28

2 4SPOI gp34

2T4 gp55

I I I I300 400 500 600

Homologies in sigma structures. The shaded section of thc bars indicate the locations ofthe highly conserved regions among the sigma factors. (This figure used with the permission of M.Gribskov.)

processes such as the heat shock response or the response to nitrogen limitation.The question remains, however: how does the cell control which 0- is used?Several strategies can be imagined, some of which are more appropriate forprogramming transient changes and some for irreversible changes in geneexpression.

Transient changes in gene expression require that the activity or amount ofthe alternate sigma be regulated in response to environmental conditions.Among possible mechanisms are these:

1. The sigma level could be changed by altering its synthesis or degradationin response to an appropriate signal. Changes in relative gene expression wouldreflect changes in the ratio of the two sigmas. Preliminary evidence suggeststhat increased amounts of 0-32 may account for the increased expression of heatshock proteins under some conditions (A. Grossman and D. Straus, un-published observation).

2. The amount or activity of additional transcription factors working at thelevel of the regulated promoters could change in response to environmentalconditions. These changes would change the effectiveness of an alternativesigma factor. Such a mechanism has been suggested for activation of thenitrogen-limited genes (S. Kustu, personal communication) and for the activa-tion of 0-37 (54) and 0-28 (42).

Sequential, irreversible changes in gene expression could be accomplished


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by successive loss of sigma factors. Among the possible mechanisms are thefollowing:

1. The preexisting ¢r factors could be displaced by newly synthesizedwith higher affinity for core RNA polymerase. Such sigma factors would haveto be synthesized in amounts at least equimolar with existing core RNApolymerase. This may be the mechanism by which 0°29 displaces previoussigma factors (R. Losick, personal communication). Chelm et al (14) comparedthe ability of phage SP01 00gp28 and B. subtilis 0043 to compete for core RNApolymerase. They found that 00gp28 can effectively compete with ~r43 for bindingto core. They concluded, however, that the degree of competition found wasnot sufficient to account for the shutoff of early transcription, and that otherfactors must be involved in the switch from early transcription by Eo"43 tOmiddle transcription by Eo"gp28.

2. The activity of preexisting 00 factors could be controlled by sigma-specificinhibitors synthesized as part of the developmental process. This mechanism isutilized by T4 to prevent 0070 function. A 10-kd protein, which is first syn-thesized starting 10 nfin after infection, antagonizes 0070, probably by binding to

it (132).3. Alternate sigma factors may be unstable. Turning off the synthesis of an

unstable sigma factor would cause its level to decrease and thereby increase theamount of core available to other sigmas. ~r29 is unstable (140-142) and itsreplacement by a subsequent 00 factor later in sporulation may depend on itsinstability.

4. Two sigma factors may bind to core simultaneously but only one woulddirect the template activity. This may be true for the T4 sigma factor 00gv55. It isknown that O"gp55 can bind to holoenzyme containing cr7° (66, 116), but it is notknown whether this results in displacement of 007o.

Role of Sigma in Promoter Recognition

Although the sigma subunit is required for specific binding of RNA polymeraseholoenzyme at promoter sequences, how sigma confers specificity is unknown.Losick, Pero, and coworkers (72a, 77, 111) suggested that each sigma confersspecificity by making contacts with both regions of the promoter. This modelwas based on the observation in B. subtilis that the promoters recognized byE0043 differ from promoters recognized by polymerases with phage or alternatebacterial ~rs at both the -10 and -35 regions (72a; reviewed in 77). Pero Losick also considered the possibility that each ~r interacts directly only at the-10 region and affects recognition at the -35 region indirectly by inducing anovel conformation in core RNA polymerase. They thought this second modelless likely; it seemed unlikely that each of the large number of sigma factorspotentially available would confer a novel conformation.

Sequence comparison ofE. coli 0070 and 003z and B. subtilis 0-43 with a set of


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380 REZNIKOFF ET AL

DNA binding proteins has identified two possible DNA binding regions in theC-terminus of the sigma factors (45, 47, 72, 155). The existence of two DNAbinding domains would be consistent with the interaction of sigmas with bothregions of the promoters. The fact that 0-70 crosslinks at both regions of thepromoter (15, 16, 129) is also consistent with this model. However, the¯ crosslinks in the -35 region have only been obtained with partially depurinatedDNA, so these results may not be biologically relevant.

The following observations, on the other hand, would be consistent withsigma factors recognizing only the - 10 region of the promoter:

1. The promoters recognized by E. coli Eo32 and Eo7° differ dramatically inthe -10 region but are similar in the -35 region (20).

2. B. subtilis 0-29 has only one region resembling the DNA binding region ofDNA binding proteins (47, 133).

3. crgp55 from E. coli phage T4 recognizes promoters that have a consensussequence only in the -10 region of the promoter (31).

4. The promoters recognized by the ntrA gene product lack a conserved - 35region, having instead two conserved regions centered around -10 and -20and separated by 3 base pairs (27, 59, 119). Because of these observations,Cowing et al (20) recently proposed a new version of the class of models which -35 region contacts are made by the core subunits of holoenzyme, notby ~r. In this model, each 0- confers specificity to holoenzyme by interactingdirectly with the - 10 region. In’addition, the different size and shape of each 0-factor could alter the precise region of holoenzyme contacting the - 35 region.This altered geometry of the holoenzyme-DNA complex could lead to dif-ferences in the spacing between the conserved sequences and in the sequence inthe -35 region recognized by holoenzyme.

According to the model, the - 10 regions of consensus promoters should besufficiently different to account for the discrimination by various forms ofholoenzyme. The -35 regions, recognized by subunits common to eachholoenzyme, could be more similar than the -10 regions recognized bydifferent sigmas, but such similarity is not required. Analysis of the availableconsensus sequences indicate that in general they conform to these expectations(Table 1). The consensus - 10 regions of promoters recognized by alternateforms of holoenzyme all lack the highly conserved TA---T sequencecharacteristic of E0-70 and Ecr43 promoters, which makes it unlikely that E0-70

could interact with these sequences. The sequences in the -35 regions are moresimilar to the conserved TTG bases in the -35 regions of Eo"7° and Eo"43

promoters.It is clear that comparison of consensus sequences recognized by alternate

forms of holoenzyme cannot settle the issue of which DNA sequences arerecognized by sigma. Additional approaches, such as mutational analysis of


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various sigmas and crosslinking studies to the closed complex, are needed to

answer this question.

ACKNOWLEDGMENTS

We wish to acknowledge the present and past members of our laboratories whohave contributed to our understanding of transcription initiation. The research

in our laboratories was supported by NIH grant GM-19670 and a 3M Founda-

tion grant to W.S.R. and NIH grants AI 19635 and GM 28575 to C.A.G.

C.A.G. was also a recipient of an NIH research career development award (AI

00573).

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the regulation of transcription initiation in bacteria...that is, the frequency of transcription...

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