Review
DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission1

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Abstract

Prokaryotic transcriptional activation often involves the formation of DNA microloops upstream of the polymerase binding site. There is substantial evidence that these microloops function to bring activator and polymerase into close spatial proximity. However additional functions are suggested by the ability of certain activators, of which FIS is the best characterised example, to facilitate polymerase binding, promoter opening and polymerase escape. We review here the evidence for the concept that the topology of the microloop formed by such activators is tightly coupled to the structural transitions in DNA mediated by RNA polymerase. In this process, which we term torsional transmission, a major function of the activator is to act as a local topological homeostat. We argue that the same mechanism may also be employed in site-specific DNA inversion.

Introduction

DNA untwisting is a crucial step in recombination and in the initiation of transcription and DNA replication. At the simplest level this can be achieved by the binding of a protein dimer which then acts as a torque-wrench untwisting the DNA between the half-sites (Figure 1A). A good example is provided by the MerR protein. Here untwisting is dependent on the binding of an effector, Hg2+, which triggers a conformational change in the protein (Ansari et al., 1992). Other examples include the restriction enzyme EcoR1 (Lesser et al., 1993) and certain mutants of the Gin invertase Klippel et al 1993, Merker et al 1993. In this simple scenario the protein on binding isolates a separate topological DNA domain distinct from the flanking regions. However the DNA topology of many of the protein assemblies that catalyse recombination, transcription initiation or replication initiation is more complex and often involves sequential topological transitions Amouyal and Buc 1987, Muskhelishvili et al 1997.

It is well established that large nucleoprotein complexes can constrain a distinct local DNA topology (Saucier & Wang, 1972) but the distribution of this constraint within a complex is often ill defined. We shall argue that not only does the DNA bound within the complex constitute a topological entity largely independent of the flanking DNA but also that functionally distinct topological domains exist within the complex itself. These microdomains, which in principle can be as short as one duplex turn as in the MerR-DNA complex, are delimited to a greater or lesser degree by protein-DNA contacts within the complex and in some cases take the form of short DNA loops (microloops) of 50 to 100 bp in extent (Figure 1B). The separation of different topological domains within an enzymatically active complex allows the possibility that the protein components could mediate topological coupling, or torsional transmission, between the domains. Here we review the evidence for this concept and note that this mode of topological coupling is distinct from that described by Bowater et al. (1994) in which the diffusion of superhelical tension generated by the processive progression of RNA polymerase directly affects the activity of a nearby promoter by altering the superhelical density of a relatively large DNA domain. By contrast, topological coupling mediated by distinct microdomains within an enzymatically active large complex allows the torsional strain to be released in discrete steps and used for overriding subsequent kinetic barriers in the reaction. This process is thus formally analogous to the stepwise release of energy in catabolic enzymatic reactions.

Section snippets

Prokaryotic transcription initiation

The evidence for torsional transmission is derived principally from studies of transcription initiation at prokaryotic stable RNA promoters (Muskhelishvili & Travers, 1997). However the paradigm for the dissection of the initiation process is the lac promoter (Figure 2A). Several distinct phases in the pathway of transcription initiation at this promoter by the σ70 holoenzyme have been distinguished (Buc & McClure, 1985). An initial rapid recognition of the −35 region is characterised by an

Stable RNA promoters

The stable RNA promoters are, as a class, among the most active promoters in Escherichia coli. Yet the core promoter region normally contains at least three deviations from the consensus structure (Lamond & Travers, 1985a). The −35 hexamer is often suboptimal with respect to the “consensus” sequence while many of the strongest promoters have a suboptimal spacer of 16 bp (Figure 3). In addition most of these promoters contain a G+C-rich region between the −10 hexamer and the transcription

Mechanism of transcriptional activation by FIS

What role do the multiple FIS binding sites play in transcription initiation at stable RNA promoters? There is substantial in vitro evidence that at both the rrnB P1 and tyrT promoters FIS recruits RNA polymerase into an initial complex and thus increases KB Bokal et al 1995, Muskhelishvili et al 1995, Muskhelishvili et al 1997. However whereas site I is apparently sufficient in vitro for this process at rrnB P1, at tyrT an insertion mutation of 5 bp which disrupts the helical phasing of the

Role of DNA geometry

The importance of DNA geometry in a different promoter context has been inferred by Hirota & Ohyama (1995) who demonstrated a preference for a right-handed writhe at certain stages of the initiation process. They showed that putative right-handed curves placed upstream of the −35 hexamer of the bla promoter were more effective than plane curves in promoting initial complex formation and promoter opening. By contrast left-handed curves repressed transcription. In this context we note that the

The role of DNA superhelix density and promoter design

We have argued that the function of FIS in a transcriptional context is to overcome impediments to initiation that reduce the overall rate of polymerase turnover at stable RNA promoters. This leaves unanswered the question of the nature of the normal physiological block. Recent evidence suggests that a major role of FIS in vivo is to act as a topological “homeostat” (Schneider et al., 1997). FIS production is maximal at the transition from stationary to exponential phase Ball et al 1992,

The activation by FIS of different steps in the initiation process

A further question is which steps in the initiation process are facilitated by FIS in vivo. The absolute level of transcription from the plasmid-borne wild-type tyrT promoter is essentially fis-independent in vivo (Lazarus & Travers, 1993). However down mutations in the −10 hexamer of both tyrT and tufB promoters confer fis-dependence (Lazarus and Travers, 1993) as also do “up” mutations in the −35 hexamer, the −35 to −10 spacer and the discriminator (A. Deufel, H. Auner, L. Lazarus, A.T. &

Other sigma70-dependent promoters

We have proposed that the UAS of stable RNA promoters functions by forming a microloop which acts as a torsional store for driving promoter opening and polymerase escape. In this section we shall address the question of whether this mode of activation by torsional transmission is peculiar to stable RNA promoters or is a particular adaptation of a mechanism that is utilised by most, or perhaps all, σ70-dependent promoters.

We make the assumption that the mechanistic aspects of core polymerase

Mechanisms of activation by transcription factors

The recruitment of RNA polymerase by a DNA-bound activator or activator complex is a straightforward and often cooperative interaction between the two components in which the factor provides an extended binding site for the polymerase either by protein-protein contacts or by bringing upstream DNA into close proximity with a secondary DNA binding site on RNA polymerase. By contrast the mechanisms by which transcription factors can facilitate subsequent steps in the initiation process are

Transcriptional activation of holoenzymes containing alternative sigma factors

So far we have considered σ70-dependent initiation. It is reasonable to ask to what extent initiation directed by other sigma factors is compatible with the concept of torsional transmission.

One particularly instructive example is provided by the late genes of E. coli bacteriophage T4. These are transcribed from a very simple promoter, consisting of a single recognition sequence TATAAATA, centred at −10 relative to the transcription start. This sequence is recognised by a phage-encoded sigma

The role of sigma factors

We have argued that the detailed mechanism by which torsion is generated and utilized differs at σ70 and σ54-dependent promoters. For the most active σ70-dependent promoters we suggest that the torsion generated by a conformational change in the holoenzyme is stored in a DNA microloop and can be subsequently utilized to drive promoter opening. By contrast at σ54-dependent promoters the equivalent conformational transition in polymerase is driven by activator-dependent NTP hydrolysis, presumably

Mechanistic parallels between transcription initiation and DNA inversion

In addition to facilitating transcription initiation FIS is also an essential component of the so-called synaptic complex which supports the site-specific DNA inversion reactions catalysed by closely related Gin, Hin, Cin and Pin invertases (for a review see van de Putte & Goosen, 1992). Are the roles of FIS in these two disparate nucleoprotein complexes mechanistically related? In common with stable RNA transcription, the efficiency of DNA inversion strongly depends on the superhelical density

The generation of torsion

The mechanism of generation of torsion can be best explained on the example of the microloops constrained by FIS. For both FIS-dependent DNA inversion and promoter opening we postulate that an essential requirement for torsional transmission is a repartitioning of twist and writhe within the microloop stabilised by FIS. Such a change in the geometry of the microloop implies a conformational flexibility which can be achieved if either the contacts between FIS and DNA or between the FIS dimers

Conclusion

This review represents an attempt to unify the great diversity of mechanisms of prokaryotic transcriptional activation by introduction of the concept of torsional transmission. This concept puts forward the view that in most, if not all systems described so far, physical coupling between proteins and between proteins and DNA in enzymatically active large complexes results in the formation of topologically closed domains which are capable of storing and utilising torsion. The stored torsion is

Acknowledgements

We thank all our colleagues who have contributed to this work during the past years. We also thank Dr G. Glaser and Dr Malcolm Buckle for communicating unpublished results. This work was in part supported by the Deutsche Forschungsgemeinschaft through SFB 190.

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