Journal of Molecular Biology
ReviewDNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission1
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.
References (118)
- et al.
Topological unwinding by RNA polymerase of strong and weak promotersa comparison between the lac wild-type and the UV5 sites of Escherichia coli
J. Mol. Biol.
(1987) - et al.
Promoter recognition by E. coli RNA polymerase. Role of spacer DNA in functional complex formation
J. Mol. Biol.
(1989) - et al.
Sequence determinants for promoter strength in the leuV operon of Escherichia coli
Gene
(1988) - et al.
Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination
J. Mol. Biol.
(1996) - et al.
Activation of transcription of σ54-dependent promoters on linear templates requires intrinsic or induced bending of DNA
J. Mol. Biol.
(1996) - et al.
Processive recombination by wild-type Gin and an enhancer-independent mutant. Insight into the mechanisms of recombination selectivity and strand exchange
J. Mol. Biol.
(1994) - et al.
Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements
J. Mol. Biol.
(1994) - et al.
Context-dependent effects of upstream A-tracts. Stimulation or inhibition of upstream promoter function
J. Mol. Biol.
(1994) - et al.
Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter
J. Mol. Biol.
(1991) - et al.
Integration host factor stimulates the phage lambda pL promoter
J. Mol. Biol.
(1990)
Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage λ pL promoter
J. Mol. Biol.
DNA determinants for rRNA synthesis in E. coligrowth rate dependent regulation, feedback inhibition, upstream activation, antitermination
Cell
Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin
J. Mol. Biol.
The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion
Curr. Biol.
The integration host factor stimulates the interaction of RNA polymerase with NIFA, the transcriptional activator of nitrogen fixation operons
Cell
Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of the bacteriophage P1. The role of FIS protein
J. Mol. Biol.
Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer
Cell
G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor
Cell
Processive recombination by the phage Mu Gin systemimplications for the mechanisms of DNA strand exchange, DNA site alignment, and enhancer action
Cell
Gin-mediated, recombination of catenated and knotted DNA substratesimplications for the mechanism of interaction between cis-acting sites
Cell
Stringent control of bacterial transcription
Cell
Genetically separable functional elements mediate the optimal expression and stringent regulation of a bacterial tRNA gene
Cell
The role of negative supercoiling in Hin-mediated site-specific recombination
J. Biol. Chem.
Crystal structure of a sigma70 subunit fragment from E. coli RNA polymerase
Cell
Factor-independent activation of Escherichia coli rRNA transcription. II. Characterisation of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase
J. Mol. Biol.
Variable structures of FIS-DNA complexes determined by flanking DNA-protein contacts
J. Mol. Biol.
Factor independent activation of rrnB P1. An extended promoter with an upstream element that dramatically increases promoter strength
J. Mol. Biol.
Action at a distanceDNA-looping and initiation of transcription
Trends Biochem. Sci.
Transcription activation via DNA-loopingvisualisation of intermediates in the activation pathway of E. coli RNA polymerase. σ54 holoenzyme by scanning force microscopy
J. Mol. Biol.
Coupling of late transcription to viral replication in bacteriophage T4 development
J. Mol. Biol.
Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-λPR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening
J. Mol. Biol.
Mechanisms of upstream activation of the rrnD promoter P1 of Escherichia coli
J. Biol. Chem.
Site-specific recombination by Tn3 resolvasetopological changes in the forward and reverse reaction
Cell
Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR
Nature
Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli
J. Bacteriol.
The isolated catalytic domain of NIFA, a bacterial enhancer-binding protein, activates transcription in vitroactivation is inhibited by NIFL
Proc. Natl Acad. Sci. USA
Effect of superhelicity on the transcription from the tet promoter of pBR322. Abortive initiation and unwinding experiments
Nucl. Acids Res.
The transcriptional activator protein FISDNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter
J. Mol. Biol.
Molecular anatomy of a transcription activation patchFIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter
EMBO J.
All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling
J. Mol. Biol.
Modulation of tyrT promoter activity by template supercoiling in vivo
EMBO J.
Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli
EMBO J.
Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps
Biochemistry
DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen
EMBO J.
Two compounds implicated in the function of the RC gene of Escherichia coli
Nature
Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli
Proc. Natl Acad. Sci. USA
Stimulation of DNA inversion by FISevidence for enhancer-independent contacts with the Gin-gix complex
Nucl. Acids Res.
Influence of DNA geometry on transcriptional activation in Escherichia coli
EMBO J.
Negative supercoiling induces spontaneous unwinding of a bacterial promoter
EMBO J.
Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniaehomologies to other nitrogen regulatory proteins
EMBO J.
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