First, we briefly describe how researchers have tried to understand the function of DNA conformation. We then detail how curved DNA is implicated in the transcription cascade.

The first functional analysis of DNA curvature was performed in 1984 using a promoter of a tRNA operon of Salmonella.³⁸ A 3 bp deletion at position -70 disrupted a curved structure, and reduced in vivo transcription to 40%. This study suggested that curved DNA could control transcription. The E. coli argT promoter requires an upstream region for high in vivo activity. Deletion mutants were used to study this promoter,⁴⁵ which required an upstream curved DNA region for high activity. Techniques using synthetic bent DNA are sometimes very useful, and have suggested that a DNA curvature close to the -35 hexamer is important.⁶³ Furthermore, linker scanning mutations were used to study the DNA curvature just upstream of the -35 sequence of E. coli rrnB operon. They revealed that the angular orientation of the DNA curvature determines promoter activation.³⁹

Insertion of a short DNA segment(s) into sites in or around the curved DNA region is also useful. In 1989, McAllister and Achberger investigated the function of curved DNA upstream of the Alu 156 promoter of B. subtilis phage SP82.⁴² By introducing short DNA fragments (6 to 29 bp) between the core promoter and the curved DNA, they changed the rotational phase between them. These changes correlated with the changes in promoter function in vivo. The most efficient mutant promoters contained insertions of 11 and 21 base pairs, and the least efficient promoters contained insertions of 15 and 25 base pairs. In vitro these mutations influenced the efficiency of RNA polymerase binding to the promoter. These findings demonstrate that the rotational phase between core promoter and the curved DNA is significant. The same methodology was used to study the promoter of C. perfringens plc gene and it was shown that upstream curved DNA stimulates transcription both in vivo and in vitro.⁴³

In 1994, using deletion mutants, Pérez-Martín and Espinosa showed that curved DNA increases transcription from the P_ctII promoter of pLS1 in vivo and in vitro, apparently independently of any activator protein.⁵⁰ Furthermore, an upstream curved DNA was replaced by the target sequence of IHF (integration host factor) or that of CopG (both are DNA-bending proteins), which activated transcription in the presence of these proteins but did not in their absence or deficiency. This study indicated that the curved conformation of DNA increases the number of contacts between the RNA polymerase and the promoter DNA, and that this increase is important in transcription initiation.

In order to alter shapes of DNA curvatures per se with minimal changes to sequence, short DNA segments were inserted into the center region of curved DNA in the pUC19 b-lactamase gene promoter,⁴⁰ and into the cyanobacterium M. aeruginosa rpoD1 gene promoter.⁴⁴ The resulting promoters were less active than the wild-type promoters, indicating that activity depended on the gross geometry. These promoters have right-handed curved DNA. Such DNAs are often located just upstream of promoters,⁶⁴ but promoter DNA itself wraps around RNAP left-handedly (fig. 3).^65-67 Thus it is tempting to speculate that if RNAP changed the writhe of the helical axis from right-handed to left-handed, it might deform the DNA and lead to local unwinding (and formation of an open promoter complex).^64,65 To test this hypothesis, right-handed curved DNA, left-handed curved DNA, two-dimensionally curved DNA and straight DNA segments were synthesized and tested for their effect on transcription in vivo. Right-handed curved DNAs clearly facilitated formation of the open promoter complex and activated transcription.⁴¹ Curved DNA can also change its shape depending on temperature.⁶⁸ Thus, temperature can be used to study the relationship between DNA conformation and function.^69,70 Recently, a mechanism of transcriptional regulation has been proposed that depends on a temperature-induced conformational change (Chapter 4).

Transcription initiation involves several steps.^71,72 Briefly, an RNAP binds to promoter (P), to yield RNAP-promoter closed complex (RPC) with an equilibrium constant K_B. Next, RNAP melts approximately 14 bp of promoter DNA surrounding the transcription start site, with a rate constant k₂, to yield an RNAP-promoter open complex (RP_O). Subsequently, RNAP begins to synthesize RNA as an RNAP-promoter initial transcribing complex (RP_ITC). There are several abortive cycles of RNA synthesis, which yield RNA of 2~8 nucleotides long, with a rate constant k_i. When by chance a 9 nucleotide strand is synthesized, RNAP releases the promoter DNA (promoter clearance) and synthesizes RNA as an RNAP-DNA elongation complex (RD_E). Bent DNA is implicated in the steps described below.

Several studies have indicated that curved DNA is important for binding RNAP to the promoter.^70,73-75 In the gal P1 promoter, a point mutation from GC to TA at position -19, which abolishes P2 activity, enhances contact between the E. coli RNAP and the DNA between -49 and -54, and activates transcription from the P1 promoter, even in the absence of cAMP-CAP.⁷³ This mutation generates a run of six consecutive thymines (5'-TTTTTT-3' is formed from 5'-TTTGTT-3'), which presumably influences the helical trajectory of the promoter, and helps RNAP binding. Hybrid E. colil phage promoters (lp_R) have been created, carrying curved DNA from the B. subtilis phage SP82 promoter, Alu156, or Bal129, immediately upstream of the -35 region. These promoters bound E. coli RNAP more efficiently than did the original promoter. Interestingly, the upstream curved DNA wrapped around the RNAP in a nucleosomal-DNA-like fashion.⁷⁵

The interaction between the a-subunit of RNAP and the curved DNA next to the core promoter may be important for initiating transcription. Many genes have an AT-rich upstream (UP) element upstream of the -35 hexamer, and this was originally identified in the E. coli rrnB P1 promoter. This element stimulates transcription through contact with the C-terminal domain of the a-subunit (a-CTD) of RNAP.^76-79 The consensus sequence of the UP element is N₂A₃(A/T)(A/T)T(A/T)T₄N₂A₄N₃.⁸⁰ This region behaved slightly abnormally during electrophoresis, suggesting that it may be curved. The curved conformation may influence interactions between the UP element and a-CTD.⁵⁷ A study using the E. coli lacUV5 promoter revealed that in the RNAP-promoter open complex, a-CTD makes alternative nonspecific interactions with the DNA minor grooves at positions -43, -53, -63, -73, -83, and -93.⁸¹ Thus around the UP element, the conformation of DNA may assist the DNA to interact with a-CTD efficiently, and as a result, may influence transcription. However, it is difficult to distinguish the effects due to the shape of DNA from effects due to an AT-rich DNA sequence per se.^78,82 None of the above studies paid attention to the 3D architecture of DNA. Interestingly, an in vitro experiment indicated that in the step of binding, E. coli RNAP favors DNA with a right-handed superhelical writhe.⁴¹

After the RNAP holoenzyme binds to the promoter, approximately 14 bp of the promoter (between positions -11 and +3) melts.⁷² An early study hypothesized that DNA curvature allows upstream DNA to contact the promoter-bound RNAP, and that these interactions help the DNA for formation of the open promoter complex.⁴² Indeed, in some cases, curved DNA enhances formation of the open complex.^41,54,83 For example, an in vitro experiment indicated that in the E. coli gal P1 promoter, the upstream curved DNA activates transcription by enhancing the rate of isomerization from the closed complex to the open complex (k₂) at P1, both in the absence of the cAMP-CAP and in its presence (the study used mutants where the P2 promoter was silenced).⁵⁴

DNA architectures are not necessarily optimally oriented towards the surface of the RNAP. The RNAP must therefore distort the DNA.⁸⁴ Actually, the promoter DNA becomes bent when RNAP binds to it, and it wraps around the enzyme with a left-handed superhelical conformation (fig. 3).^65-67,85,86 Energy invested in DNA bending could open the double helix.⁸⁷ These considerations suggest that DNA curvatures can enhance stress, depending on the direction of the curvature, which explains well why many bacterial promoters have a right-handed curved DNA immediately upstream of the -35 region.⁶⁴ This right-handed curvature presumably increases stress when the DNA wraps around RNAP, reversing its superhelical handedness. Indeed, when a synthetic right-handed curved DNA segment was placed immediately upstream of the -35 region of pUC19 b-lactamase promoter, it facilitated DNA melting at positions -11, -9, and +3 and activated transcription, compared to left-handed curved, two-dimensionally curved, and zigzag (straight) DNA segments.⁴¹ Positions -11 and +3 correspond to the limits of RNAP-induced promoter melting.⁷² Based on this putative mechanism, modulating the shape of DNA can also produce negative effects. A T₅ tract introduced just upstream of the -35 hexamer reduced promoter activity.⁸⁸ As another example, curved DNA inhibited transcription from the B. subtilis P_A2b promoter, by reducing the ability of RNAP to form transcriptionally active open complexes (in this case, binding of RNAP to the promoter was also impaired).⁸⁹ However, it is still not clear why DNA curvatures which are preset to modulate promoter melting, are usually located upstream of the core promoter.

In the last stage of transcription initiation, promoter escape or promoter clearance occurs. "Escape" deals directly with issues affecting the downstream movement of a polymerase molecule. On the other hand, "clearance" implies that the polymerase moves far enough downstream to make the core promoter available to a second polymerase.^90,91 These phenomena are also rate-limiting steps in transcription initiation. In the B. subtilis page F29 A2c promoter, preventing RNAP escape represses transcription.⁹² Are DNA curvatures involved in promoter escape or promoter clearance? The following study suggests they are. In a study using two promoters isolated from the B. subtilis bacteriophage SP82, curved DNA with a strong affinity for RNAP reduced transcription, while curved DNA with a weaker affinity stimulated transcription. ⁷⁴ DNA curvatures may collaborate with H-NS to reduce promoter clearance - it is known that they can repress transcription (Chapter 4). Images from scanning force microscopy (SFM) showed that in an E. coli rrnB P1 promoter with an upstream DNA curvature, H-NS trapped RNA polymerase in the open initiation complex. The SFM images suggested that H-NS-mediated trapping of RNAP could prevent promoter clearance.⁹³ To the best of our knowledge, a positive influence of DNA curvature has not been reported.

If a curved DNA overlaps with some cis-DNA element, the curve's role may be to recruit trans-acting factors. In the M. aeruginosa psbA promoter, the curved region (approximately -180 to -140) is bound by a protein factor, and mutants having altered curvature had decreased basal transcription.⁴⁸E. coli OmpR and B. subtilis Spo0A are response regulators in bacterial two-component regulatory systems.⁹⁴ Although OmpR can stimulate transcription of ompF (at low osmotic strength), and of ompC (at high osmotic strength), the OmpR binding site in the ompC promoter seems not to form a curved DNA. Thus, the presence or absence of curved DNA may be distinguished by OmpR in the differential activation of ompF and ompC promoters.⁵⁵ The B. subtilis spo0F gene, which decides the cell fate in sporulation initiation, has a tandem promoter, P1 and P2. P1 is recognized by s^A-RNAP during exponential cell growth, and P2 is recognized by s^H-RNAP during initiation of sporulation. For Spo0A binding at P1, the center of DNA curvature is close to the 0A-boxes. Increased Spo0A binding to the 0A-boxes represses transcription from the upstream P1, and simultaneously induces transcription from the downstream P2.³² Curved DNA may facilitate Spo0A binding. However, no report has shown conclusively that curved DNA conformation per se is involved in factor binding.

Intrinsically curved DNA is sometimes located between activator binding sites and promoters. Several examples indicate that these structures enable activator and RNAP to interact. In the s^N-dependent glnAp2 or glnHp2 promoter of E. coli, the DNA between the NR_I (NtrC)-binding site and the s^N-recognition region must be either intrinsically curved, or curved by binding of the integration host factor, IHF. The glnAp2 has an intrinsic DNA curvature in the relevant region. Although glnHp2 does not carry curved DNA, it has an IHF-binding site in the relevant region. The DNA bend allows the activator to contact the s^N-RNAP-promoter complex. This activates transcription by catalyzing the isomerization of the closed s^N-RNAP-promoter complex to form an open complex.^59,95,96

However, when supercoiled DNA templates are used, transcription can be initiated even in the absence of such a bend,⁹⁶ meaning that the spatial alignment between NR_I and RNAP on the templates may be significant. Similarly, in the K. pneumoniae nifLA promoter, an activator protein NtrC may interact with the s^N-RNAP holoenzyme bound to the promoter with the help of an intervening curved DNA.⁶⁰ As another example, in the E. coli rrnB P1 promoter, the rotational phase between the FIS binding site and the promoter is important in activating transcription.⁹⁷ The curvature between these regions may also help FIS and RNAP to spatially position themselves correctly.

Curved DNA can also play a role in packaging of genomes. In eukaryotes, this role is played in nucleosome or chromatin formation (see Chapter 13). In E. coli, some nucleoid-associated proteins, such as H-NS (histone-like nucleoid-structuring protein), CbpA (curved DNA-binding protein A), Hfq (host factor for phage Q_b), and IciA (inhibitor of chromosome initiation A) preferentially bind to curved DNA.^98-100 Of these, H-NS can condense DNA both in vitro and in vivo^101,102 and regulates expression of a number of genes,^26,103 which are described in Chapter 4.

Curved DNA may also modulate the geometry of promoters in collaboration with structure-specific transcriptional regulators. The B. subtilis LrpC protein, which belongs to the LrpC/AsnC family of transcriptional regulators, forms stable complexes with curved DNAs. Interestingly, LrpC proteins wrap DNA to form nucleosome-like structures (but containing positively supercoiled DNA), and it is speculated that these could regulate transcription.¹⁰⁴ As another function, temperature-dependent conformational changes of bent DNA, as observed for M. aeruginosa psbA2 gene,⁴⁷ can regulate transcription of several genes (Chapter 4).