INTRODUCTION

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The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing growth regimens. Before they can transcribe from specific DNA promoter sequences, bacterial core RNA polymerases (with subunit composition ₂') must combine with a dissociable sigma subunit () to form RNA polymerase holoenzyme (₂'). Since the discovery of  factors (6), it has become clear that these proteins are central to the function of the RNA polymerase holoenzyme. The reversible binding of alternative  factors allows formation of different holoenzymes able to distinguish groups of promoters required for different cellular functions. In addition to double-strand DNA promoter recognition and binding,  proteins are closely involved in promoter melting (e.g., references 31, 36, 49, 51, 74, 76, 128), inhibit nonspecific initiation, are targets for activators, and control early transcription through promoter clearance and release from RNA polymerase (48, 49, 53). Here we describe the functioning of the bacterial ⁵⁴-RNA polymerase that is the target for sophisticated signal transduction pathways (103) involving activation via remote enhancer elements (5, 95).

Based on structural and functional criteria, the different  factors identified in bacteria can be grouped in two classes, one of which has a single member, ⁵⁴. Many  factors belong to the ⁷⁰ class, the major  factor which is involved in expression of most genes during exponential growth (72). ⁵⁴ (also called ^N) differs both in amino acid sequence and in transcription mechanism from the ⁷⁰ class (80). Despite the lack of any significant sequence similarity, both types of  bind the same core RNA polymerase. Nonetheless, they produce holoenzymes with different properties.

With the recognition that the ⁵⁴ protein represented an entirely new class of  factor, what had once been regarded as an aspect of transcription restricted to higher organisms became a well-established feature of certain bacterial regulatory systems, particularly those associated with nitrogen metabolism. Activation of ⁵⁴-RNA polymerase employs specialized bacterial enhancer-binding proteins whose activating function requires nucleotide hydrolysis (94, 96, 122) (Fig. 1). In this system, initiation rates are controlled via regulation of the DNA melting step that is necessary for establishing the open promoter complex (85, 94, 97). Bacterial enhancer-dependent transcription can be studied with just two purified proteins (an activator and the ⁵⁴-RNA polymerase holoenzyme) and the appropriate DNA template, facilitating progress in understanding mechanistic aspects of ⁵⁴ functioning. Below we review the biology and biochemistry of the ⁵⁴-RNA polymerase.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   RNA polymerase holoenzyme containing 54 binds to 24 and 12 consensus promoters to form a stable closed complex which is transcriptionally silent. This complex can be disrupted by heparin in vitro. Activator-mediated nucleotide hydrolysis drives full open promoter complex formation. Open complexes are insensitive to heparin in vitro. NTP, nucleoside triphosphate; NDP, nucleoside diphosphate.

	OCCURRENCE AND FUNCTION OF 54

Although ⁵⁴ was originally recognized in the enteric bacteria, it is now clear that ⁵⁴ is widely distributed among the bacteria. The role of ⁵⁴-RNA polymerase, historically in regulation of nitrogen metabolism and subsequently in many other biological activities, is well established in many proteobacteria (80). Genes encoding ⁵⁴ have also been cloned from the gram-positive Bacillus subtilis, where ⁵⁴ is involved in utilization of arginine and ornithine (43) and transport of fructose (29), and from Planctomyces limnophilus (68). Furthermore, genome sequencing projects have revealed open reading frames potentially encoding ⁵⁴ in diverse bacteria such as an extreme thermophile (30, 105), obligate intracellular pathogens (58, 102), spirochetes (37, 38), and green sulfur bacteria (104). However, complete genome sequences have also revealed the absence of ⁵⁴ in a diverse range of bacteria including the high-G+C gram-positive Mycobacterium tuberculosis, the extreme thermophile Thermotoga maritima, the specialized pathogens Rickettsia prowazekii, Mycoplasma genitalium, and Mycoplasma pneumoniae, the photosynthetic Synechocystis sp. strain PCC6803, and Deinococcus radiodurans (for a recent review, see reference 104).

Most bacteria contain several alternative  factors belonging to the ⁷⁰ class, but two forms of ⁵⁴ rarely coexist in the same organism. That is, no more than one ⁵⁴ gene is usually found, the exceptions so far being Bradyrhizobium japonicum, Rhodobacter sphaeroides, and Rhizobium etli which each contain two rpoN genes encoding two ⁵⁴ proteins (24, 65, 82). Nonetheless, ⁵⁴-RNA polymerase can be regulated independently at a wide variety of genes by virtue of a family of sequence-dependent enhancer proteins with promoter-specific binding sites (26). Each protein is controlled by its own signal transduction pathway, thus allowing a single ⁵⁴ polypeptide type to mediate transcriptional responses to a wide variety of physiological needs.

There is no obvious theme in the repertoire of functions carried out by the products of ⁵⁴-dependent transcription. Among the proteobacteria, these include utilization of various nitrogen and carbon sources, energy metabolism (70), RNA modification (44), chemotaxis, development, flagellation, electron transport, response to heat and phage shock (123), and expression of alternative  factors (reviewed in references 1, 66, 80, and 104). It appears that ⁵⁴ is not usually essential for survival and growth under favorable conditions, except in Myxococcus xanthus (62).

The pattern of its occurrence in the bacterial domain argues for ⁵⁴ being biologically important and advantageous. Because initiation of transcription at a ⁵⁴-dependent promoter absolutely requires the activity of the cognate activator protein, transcription can be very tightly regulated, with low levels of leaky expression (118). Moreover, the use of ⁵⁴ may confer one notable advantage: the capacity to vary transcriptional efficiency at a given promoter over a wide range without the use of a separate repressor. Genes transcribed by this form of polymerase can be silent or highly expressed (when activated), depending on the physiological or environmental conditions. Given these advantages, why then are relatively few bacterial genes transcribed by ⁵⁴-RNA polymerase? The pathogenic Neisseria spp. seem to have abandoned the ⁵⁴-RNA polymerase mode of transcription recently, their genomes still containing rpoN pseudogenes which have apparently undergone a deletion of the DNA-binding region (67). Presumably the main disadvantage of the ⁵⁴-RNA polymerase mode of transcription is the requirement for significant stretches of intergenic DNA, and thus the need for larger chromosomes. The requirement for additional intergenic DNA arises from the DNA-looping mechanism of activator-RNA polymerase contact (5, 95). In order for cross talk between transcription units to be minimized, promoters would need to be well isolated from each other (73), as occurs in higher organisms where looping is common. In the case of nif genes in Klebsiella pneumoniae and Azotobacter vinelandii, their clustering could lead to promiscuous activation of one nif operon by the NifA bound to another nif operon's DNA. However, any cross activation would still be by the same signal transduction pathway, and might therefore be readily tolerated. Therefore, the same pressures that have led to the compactness characteristic of bacterial genomes may select against the increased use of ⁵⁴-RNA polymerase.

	CONTROL CIRCUITRY

⁵⁴-dependent activators bind to DNA sites at atypically long distances (for bacteria) from the start site for transcription, consistent with the looping mechanism of activation. This mechanism coexists with those regulating activity of the ⁷⁰-holoenzyme, for which activators bind adjacent to the polymerase site and touch the enzyme without looping (45). The ⁵⁴-holoenzyme forms a closed complex and occupies the promoter in this state prior to activation (97). This closed complex is unusually stable in the sense that it does not spontaneously isomerize into an open complex. Basal, unactivated transcription from the closed complex is intrinsically very low, consistent with the lack of repressors (see reference 26 but see reference 121 also) associated with ⁵⁴-dependent promoters. This stable closed complex is a convenient target for the looping of activators bound to remote sites (106). At some promoters, the looping-out of the intervening DNA is facilitated by the bending protein integration host factor (IHF) (54). This effect can be mimicked by HU or even the mammalian nonhistone chromatin protein HMG-1 and can be bypassed by intrinsically curved DNA (13, 15, 92, 93). IHF has been proposed to stimulate recruitment of ⁵⁴-polymerase at least one promoter (2). The outcome of looping is an activator-dependent isomerization of the closed complex into an open one, which leads to initiation of transcription. This mechanism is not observed for ⁷⁰-holoenzyme, where both highly stable closed complexes and looping are rarely, if ever, used directly for activation (46). In essence, ⁵⁴ binding to RNA polymerase imposes a block on the initiation pathway whereby open-complex formation (DNA melting) can be controlled independently from closed-complex formation (DNA binding).

The use of enhancers, nucleotide hydrolysis for melting (120), and chromosome structure modification by bending are more commonly found in cases of eukaryotic polymerase II transcription than with bacterial ⁷⁰-based transcription (reviewed in reference 64). Their involvement in bacterial ⁵⁴-dependent transcription indicates that ⁵⁴ is responsible for significantly modifying the properties of the RNA polymerase to endow enhancer responsiveness. In this sense ⁵⁴ converts the polymerase to an enhancer-requiring enzyme (115). The mechanisms used by eukaryotic enhancers are not limited to the melting control observed in bacteria. The need for compactness in bacterial genomes may preclude the greater diversity of mechanisms that occur in mammalian cells.

As is the case for ⁷⁰-holoenzyme, activation of ⁵⁴-RNA polymerase occurs by signal transduction pathways using numerous and diverse activators (reviewed in reference 101). Although these pathways are diverse, they have a common terminal mechanism. In each case, the output appears to involve the triggering of a hidden ATPase activity within an enhancer-binding activator protein. This ATPase is then used to overcome the block to DNA melting within the closed transcription complex and thereby allow transcription to initiate (122). When the physiological stimulus is removed, the activators no longer have their ATPase activities triggered and the cognate promoters revert to the inactive closed complex state. Many activators have their ATPase activities triggered by a phosphorylation cascade, as typified by the nitrogen regulator NtrC (124). Changing physiological conditions typically leads to phosphorylation of the protein's N terminus. Conformational changes then can lead to changes in activator affinity for enhancer DNA sites, multimerization on the DNA, and most important, assembly of a DNA-bound complex with ATPase activity (96). The ATPase activity is within the central domain of the activator, which may adopt a fold common to other purine nucleotide-binding and hydrolyzing proteins, and is predicted to show some structural similarity to members of the AAA+ protein family to which it belongs (87). Our understanding of NtrC is increasing with knowledge of its N- and C-terminal domain structures and how its activities are controlled by phosphorelay (61, 90, 91). Many other activators, e.g., NifA and PspF, do not rely on phosphorylation but instead react with small-molecule effectors or inhibitory polypeptides (e.g., references 32, 34, 101 and 129).

Additional physiological controls may be superimposed on the activator-dependent control of transcription from ⁵⁴-dependent promoters. For example, the pseudomonas putida Pu and Po promoters and the Rhizobium meliloti dct promoter are subject to catabolic repression by mechanisms which appear to be independent of signal transduction via the cognate activators (19-21, 33, 75, 83, 121). RNA polymerase holoenzyme at these promoters may also be regulated by a mechanism involving ppGpp (e.g., reference 110). Furthermore, the FtsH (HflB) protease is required for full ⁵⁴ activity in vivo at least at some promoters (14).

Although expression of ⁵⁴ is constitutive in many bacteria investigated (reviewed in reference 128), it is temporally regulated in Caulobacter crescentus (3) and Chlamydia trachomatis (77). Transcription of rpoN, encoding ⁵⁴, is probably subject to negative autoregulation in several bacteria including Acinetobacter calcoaceticus (35), Azotobacter vinelandii (79), B. japonicum (65), K. pneumoniae (47, 78), P. putida (63), and R. etli (82, 83).

The paradigm for enhancer-dependent transcription in eubacteria is characterized by interaction between ⁵⁴-RNA polymerase and an activator of the NtrC/NifA family. However, evidence is emerging that there may be more-complex assemblies of protein at some promoters controlled by ⁵⁴ that contribute to sophisticated regulatory responses. For example, cyclic AMP receptor protein mediates repression at the dctA promoter of Sinorhizobium meliloti (121).

A further example of complex control of ⁵⁴-dependent transcription may occur in B. subtilis where the AhrC protein represses arginine biosynthesis by binding to operator sites in the promoter regions of arginine biosynthetic genes (71, 84). An AhrC-binding site is located between the genes for glutamate dehydrogenase (rocG) and 1-pyrroline 5-carboxylate dehydrogenase (rocA), both of which may be under the control of ⁵⁴-RNA polymerase and the activator RocR (43). AhrC footprints a region directly adjacent to the rocA gene (nucleotides 22 to 2), suggesting the possibility of a regulatory interaction with ⁵⁴-RNA polymerase.

	54 DOMAIN STRUCTURE, PROMOTER RECOGNITION, AND CORE INTERACTIONS

Sequence alignments, mutation analyses, and protein fragmentation studies have led to a picture of the overall domain structure of ⁵⁴, mainly using the proteins from enteric bacteria (11, 40, 79, 98, 127). ⁵⁴ was divided into three regions by sequence conservation (Fig. 2) (80). Primary DNA-binding functions (127) are in region III with a DNA cross-linking region (7) and associated motifs, the helix-turn-helix motif (81) and RpoN box (111) near the C terminus. Adjacent sequences modulate this activity (12) and others constitute the minimal core-binding domain (40, 113). Region II is variable and in some species, such as Rhodobacter capsulatus, is almost completely absent. In many (but not all) bacteria, region II is acidic, and it has been implicated in DNA melting (126), in the transition from the closed to open complex (E. Southern and M. Merrick, submitted for publication), and in assisting ⁵⁴ binding to homoduplex and heteroduplex DNA (8). The lack of conservation of region II suggests that none of its activities are essential. The amino-terminal 50 residues (region I) comprise a domain that performs two distinct functions: (i) inhibiting polymerase isomerization and initiation in the absence of activation (10, 109, 115) and (ii) stimulating initiation in response to activation (98, 109). It is now clear that there is considerable cross talk among these domains for the full function of ⁵⁴.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Domain organization of the 54 protein. E. coli 54 (amino acids 1 to 477) can be divided into three regions (I to III [80]). DNA-binding functions (127) and associated motifs (DNA cross-linking [XLINK] region [7], helix-turn-helix [HTH] motif [81], and RpoN box [111]) reside in the C terminus, adjacent to sequences that modulate DNA binding (amino acids 180 to 306) (12). Activator responsiveness involves region I sequences (10, 98, 109), and region II is acid and variable. The main core binding determinant from amino acids 120 to 215 (40, 127) is shown. A full 54 amino acid alignment of known sequences is available at http://www.bio.ic.ac.uk/staff/mbuck/alignment.rtf.

DNA binding. The primary DNA-binding activity for recognition of double-stranded promoter DNA resides in a C-terminal domain (7, 50, 81, 98, 111, 127). Mutations within this region eliminate such binding. Unlike ⁷⁰, the DNA-binding activity of ⁵⁴ is not fully masked and ⁵⁴ is able to bind to certain promoters in the absence of core polymerase (4). Nonetheless, holoenzyme binds tighter than does ⁵⁴ alone. Two other regions can affect the affinity of binding: the N terminus plays complex multiple roles in binding to both duplex and melted DNA (10, 17, 41, 52, 59, 60, 98, 117), and the segment between the C terminus and the major core-binding determinant influences binding affinity (12, 50). These regions do not necessarily make direct DNA contact.

Core polymerase binding. The interface between ⁵⁴ and core RNA polymerase is probably very extensive. Mutations in the central region of ⁵⁴ eliminate binding to core (56, 112, 113, 127). Additional contributions come from the C-terminal DNA-binding domain and N-terminal region I (16, 17, 40). Although ⁵⁴ is not related to ⁷⁰ by primary sequence, it binds to the same core polymerase, likely with similar affinity (40, 99). However, there is a short region of potential similarity in the central domain (112) and small-angle X-ray-scattering studies suggest that the core-binding domains of ⁵⁴ and ⁷⁰ have similar shapes (107). Results with tethered iron chelate methodology have suggested related binding arrangements (125). The core-binding interface of ⁷⁰ also involves widely separated regions of that protein (100), yet another point of similarity between ⁵⁴ and ⁷⁰. ⁷⁰ has determinants of interaction with the core and with the DNA in close proximity. The N terminus of ⁵⁴ also has these properties, which may reflect a coordinated behavior of domains during transcription initiation.

	MECHANISM OF PROMOTER ACTIVATION

The key activation event is the opening of the DNA within the closed complex of ⁵⁴ holoenzyme at the promoter. This is accomplished using the ATPase activity of the activator, which loops from the enhancer site (96). The interactions between activator and the ⁵⁴-RNA polymerase holoenzyme appear to be transient, and the only direct physical evidence has been by a cross-linking assay using the DctD activator (60, 69).

As activators of the ⁵⁴-holoenzyme are related to the purine nucleotide-binding and hydrolyzing proteins, they would be expected to use the ATPase to bring about conformational changes. It is not known how the proposed energy coupling impacts upon the holoenzyme to cause it to open the DNA. Sequence analysis of activators does not reveal obvious similarity to known helicases (96, 122).

Clues from deregulated transcription. One view of the activation process is that ⁵⁴ organizes the holoenzyme so that it cannot fully melt DNA; the activator then overcomes this block. In this view, mutants that allow unregulated melting provide valuable clues to the mechanism of activation (115). Such mutants have been found in both the ⁵⁴ protein and in the DNA sequence of the promoter.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Cartoon model showing activities proposed for activation via DNA melting. Dark shading indicates activities primarily in 54. These activities include duplex (double-stranded DNA) binding at nucleotides 24 and 12 and fork junction binding. The 7 to 11 single-strand binding activity is seen only after activation. Light shading indicates single-strand binding activities contributed by RNA polymerase holoenzyme. The solid circle represents the 11 connector nucleotide that inhibits the spread of unactivated melting when unpaired. (Top) Prior to activation, the holoenzyme is stabilized on the DNA using primary interactions at nucleotide 24. The unactivated complex includes a form shown here in which a molecular structure near 12 contributes to binding and prevents the spread of early DNA melting to downstream positions. The N terminus and the fork junction connector position 11 (solid circle) are key determinants in locking the system in a closed state. Deregulated bypass mutations destroy this 54-DNA structure at 11 and allow partial engagement of the downstream single-strand binding activities; these are shown downstream of 12 and above and below the DNA in an unengaged state. (Bottom) Upon activation, ATP triggers conformational changes in 54 at the fork junction that involve the 54 N terminus. The upstream nontemplate strand binding activity is exposed, and interactions through the connector can now be established. The single-strand DNA-binding activities stabilize the spread of melting.

Role of the ⁵⁴ polypeptide. Various results underscore the pivotal role of ⁵⁴ in activation and DNA melting by holoenzyme and suggest that it may be the primary target of the activator. ⁵⁴ has the specificity that recognizes the DNA at the upstream fork junction (52). ⁵⁴ bound to heteroduplex DNA probes containing such junctions changes conformation independently of core polymerase in a reaction that requires activator and nucleotide hydrolysis (9). In the isomerized complex, ⁵⁴ interactions give an extended DNase I footprint that approaches the start site and some DNA melting occurs over this region, (W. Cannon, M.-T. Gallegos, and M. Buck, unpublished data). These considerations indicate that conformational changes in ⁵⁴ itself are likely to be very early events triggered by activators.

	FUTURE DIRECTIONS

Remaining problems include determining the precise sites of contact for activators in ⁵⁴-holoenzyme, working out how the activator uses ATP to achieve conformational changes, and determining what these conformational changes are and how they are used. Some answers will depend upon structural information and biophysical approaches. Genetic methods still continue to be a way forward (e.g., reference 47). We note that the ⁵⁴ transcription mechanism complements the common bacterial mechanism with a dependence on enhancers and ATP hydrolysis for initiation that is like mammalian RNA polymerase II. In a sense, this bacterial arrangement mimics the specialization in eukaryotes where different polymerases have differing dependencies on enhancers and ATP hydrolysis. It will be a challenge to work out the advantages of each type of mechanism and place them together in evolutionary context.