DNA Melting within a Binary sigma 54-Promoter DNA Complex*

Wendy CannonDagger , María-Trinidad Gallegos§, and Martin BuckDagger

From the Dagger  Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom and the § Departmento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain

Received for publication, August 25, 2000, and in revised form, October 9, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The sigma 54 subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that sigma 54 is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that sigma 54 contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that sigma 54 binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in sigma 54 to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to sigma 54 interactions and sigma 54-DNA interactions occur in response to activator to allow sigma 54 isomerization and the holoenzyme to progress from the closed complex to the open complex.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Accessing the information in DNA often relies upon the action of DNA-binding proteins that are able to generate non-canonical B-DNA structures. Recombination, replication, methylation, repair, and transcription are processes that proceed through intermediates in which DNA is distorted. The process of RNA transcript formation by all RNA polymerases must involve a DNA-melting event to reveal the template DNA strand (1-3). Distortion of the DNA leading to the nucleation of strand separation occurs within the closed complex formed between RNA polymerases and the promoter. Following isomerization of the polymerase, full stable DNA opening is evident, which is thought to have spread from the initial nucleation site. Single-stranded DNA-binding activities in the RNA polymerase are required for DNA opening, and in the case of the bacterial RNA polymerase, the sigma  subunit plays an important role (1, 3-7). For the sigma 70-type factor, binding to the core enzyme induces conformational changes in a single-stranded DNA-binding region of the protein. As a consequence of these conformational changes, sigma 70 gains specificity for the non-template strand of the melted region in the open complex (8, 9). For the sigma 54-type factor, unrelated by sequence to sigma 70, the determinants of single-stranded DNA binding are less well described. Single-stranded DNA binding by sigma 54 is evident, however (4, 10, 11). The sequences that sigma 54 recognizes as single-stranded DNA are between the -12 promoter element and the start site (4). Importantly, the activity of the sigma 54 holoenzyme is tightly regulated at the DNA-melting step, but promoter binding to form the initial sigma 54 holoenzyme closed complex is not highly regulated (12).

The closed complexes formed with the sigma 54 holoenzyme are silent for transcription unless acted upon by an enhancer-binding activator protein (13-15). A network of protein and DNA interactions involving sigma 54 function to maintain a stable holoenzyme conformation that rarely changes spontaneously to allow DNA melting and transcript initiation (11, 16-22). The conformationally restricted closed promoter complex isomerizes to an open promoter complex (in which the DNA strands are melted out) in a reaction in which the activator consumes ATP or another nucleoside triphosphate (14). As a part of this reaction pathway, sigma 54 contributes to the creation of a local structural distortion within the closed complex (23). sigma 54 binds tightly to the distorted promoter DNA and can be shown to isomerize independently of the core RNA polymerase in a reaction that has all the remaining requirements for open complex formation (4, 24). Isomerization is associated with an increased DNase I footprint of sigma 54 on DNA, extending toward the transcription start site (24).

Here we use DNA footprinting to show that the DNA within the isomerized sigma 54-DNA complex has melted and that some melting is negatively regulated by Region I of sigma 54. However, extensive melting requires Region I. Additionally, changed interactions between sigma 54 and the nucleated DNA are evident in complexes in which melting has occurred. We show that the presence of core RNA polymerase inhibits those changes in sigma 54-DNA interactions that occur in response to activator, consistent with the view that tight binding to the early melted DNA limits DNA opening (4). The results provide clear evidence in favor of an activation mechanism in which conformational changes in a basal sigma 54-DNA complex are brought about by the enhancer-binding activator. Activator-independent melting suggests that the activator does not function exclusively as a site-specific DNA helicase for DNA opening (11, 18, 22).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA and Proteins-- The promoter fragments used in this work were Escherichia coli glnHp2 from -60 to +28 in which T at -13 was replaced by G and the -11/-10 sequence was replaced with a CA/TG mismatch to create a short unpaired DNA element next to a consensus GC (-13/-12) promoter element or a mismatched sequence between -11 and -6 (see Fig. 1). Sinorhizobium meliloti nifH promoter fragments from -60 to +28 either containing a CA/TG mismatch immediately adjacent to the consensus GC element or with the A-12 top strand base missing (gapped duplex) were also used (see Fig. 1) (24). Synthetic DNA strands from -60 to +28 were annealed to create the duplex, with either strand 5'-32P-end-labeled. The unlabeled strand was at 2-fold molar excess.

The Klebsiella pneumoniae sigma 54 protein, its Region I-deleted derivative lacking the first 56 amino acids (Delta Isigma 54), and Region I (amino acids 1-56) were prepared as described previously (11, 25). The activator was E. coli PspF lacking a functional C-terminal DNA-binding domain (PspFDelta HTH) (26). E. coli core RNA polymerase was from Epicentre Technologies Corp.

DNA Binding Assays-- End-labeled DNA (16-100 nM) and 1 µM sigma 54 or Delta Isigma 54 in a 10-µl reaction in buffer containing 25 mM Tris acetate (pH 8.0), 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% (w/v) polyethylene glycol 8000 were incubated for 5 min at 30 °C. Activator PspFDelta HTH (0.5-4 µM) and dGTP, GTP, or GTPgamma S1 (4 mM) were added for a further 10 min. Region I was at 0.5 µM. Where indicated, heparin (100 µg/ml) was added for 5 min prior to gel loading. Free DNA was separated from sigma -bound DNA on 4.5% native polyacrylamide gels run in 25 mM Tris and 200 mM glycine at room temperature.

DNA Footprints-- Binding reactions were conducted as described above; footprinting reagents were added; reactions were terminated; and bound and unbound DNAs were separated on native gels as described above. DNA was then excised, processed, and analyzed on a denaturing 10% polyacrylamide gel. For DNase I footprints, 1.75 × 10-3 units of enzyme (Amersham Pharmacia Biotech) was added to a 10-µl binding reaction for 1 min, followed by addition of 10 mM EDTA to stop cutting. For KMnO4 footprinting, 4 mM fresh KMnO4 was added for 30 s, followed by 50 mM beta -mercaptoethanol to quench DNA oxidation. Gel-isolated DNA was eluted into 0.1 mM EDTA (pH 8.0) (DNase I footprints) or H2O (KMnO4 footprints) overnight at 37 °C. KMnO4-oxidized DNA was cleaved with 10% (v/v) piperidine at 90 °C for 20 min. Recoveries of isolated DNA were determined by dry Cerenkov counting, and equal numbers of counts were loaded onto gels.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, we showed that purified sigma 54 bound to the S. meliloti nifH promoter was able to isomerize if the DNA template had an unpaired sequence downstream of the GC element of the promoter (24). The isomerization also required activator and nucleoside triphosphate hydrolysis and was characterized as an extended sigma 54-DNA interaction toward the transcription start site. The unpaired DNA downstream of the GC promoter element was suggested to mimic the nucleation of DNA melting (early melted DNA) seen in sigma 54 closed complexes, which normally requires sigma 54 and core RNA polymerase (23). Here we have used variants of the sigma 54-dependent E. coli glnHp2 promoter (27) (Fig. 1) to explore DNA melting by sigma 54. We chose to generate a nucleated glnHp2 promoter because the high AT content of the sequence should facilitate the detection of unstacked T residues using KMnO4 as a DNA footprinting reagent (28). Base unstacking occurs when DNA melts, and the associated increased reactivity to KMnO4 is readily detected.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence of the E. coli glnHp2 and S. meliloti nifH derivatives used in this work. Compared with the original glnHp2 m12 promoter sequence (27), heteroduplex promoter fragments contained either unpaired DNA from -11 to -10 with A-10 (bottom strand) replaced with G to form a pre-melted structure comparable to that for the early melted S. meliloti nifH DNA (24) or from -11 to -6 (highlighted). In glnHp2 m12, the wild-type T:A base pair at -13 is replaced by G:C to increase sigma  binding. S. meliloti nifH promoter fragments are as described previously (24).

Isomerization of the sigma 54-glnHp2 Complex-- Initially, we used a gel shift assay to show that sigma 54 bound to the modified glnHp2 promoter with unpaired DNA at -11/-10 and, in a reaction requiring hydrolyzable nucleoside triphosphate (dGTP) and activator, produced a supershifted heparin-resistant complex (sssigma -DNA, Fig. 2A, lane 4). As seen before, the same mobility-supershifted complex formed with activators of different molecular weights, providing evidence that the activator was not stably associated with the isomerized complex (Ref. 24 and data not shown). Formation of this complex required sigma 54 Region I (Fig. 2A, compare lanes 4 and 8). Addition of Region I in trans to Delta Isigma 54 resulted in a new species with similar mobility to that of the supershifted complex, independent of activator and nucleotide (Fig. 2A, lanes 9 and 10). Using DNase I footprinting, we showed that the DNA within the isomerized sigma 54 complex was protected more than in the complex formed with sigma 54 in the absence of activator and nucleotide (Fig. 2B). The downstream edge of the sigma 54 footprint extended to about -5 (Fig. 2B, lane 3). In the isomerized complex, the footprint extended clearly to +2, but a partial footprint to +5 was also detected (Fig. 2B, lane 4). The upstream edge of the footprint was not easily discernible due to background DNA fragments in the undigested sample (Fig. 2B, lanes 1 and 5). Binding of Delta Isigma 54 did not lead to the extended DNase I footprint of sigma 54 seen with the activator-dependent isomerized complex, but weakly footprinted to about -7 (Fig. 2B, lanes 9 and 10). Addition of Region I in trans to Delta Isigma 54 resulted in a DNase I footprint indistinguishable from that of the non-isomerized sigma 54-DNA complex (Fig. 2B, compare lanes 7 and 8 with the lane 3).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.   The supershifted isomerized sigma 54-DNA complex (sssigma -DNA) forms in a reaction requiring activator and a hydrolyzable nucleotide. A, gel shift assays were conducted with sigma 54 or Delta Isigma 54 (1 µM), Region I (RI; 0.5 µM), end-labeled glnHp2 -11/-10 DNA (16 nM), PspFDelta HTH activator (0.5 µM), and dGTP (4 mM) where indicated. sigma -DNA complexes were challenged with heparin (100 µg/ml for 5 min) and loaded onto a native polyacrylamide gel, where bound and unbound DNAs were separated. B, the isomerized sigma 54-DNA complex has an extended DNase I footprint. Comparison of sigma 54 and isomerized complexes shows that the footprint is extended by the action of activator. Removal of Region I prevents formation of the extended footprint. Reactions (10 µl) were as described for A, except that glnHp2 -11/-10 DNA was at 50 nM, and PspFDelta HTH was at 4 µM. Following exposure to DNase I, bound and unbound DNAs were separated on a native gel, eluted, and analyzed on a sequencing gel. Additions to each binding reaction are shown above each lane. Lanes 1 and 5, untreated DNA; lanes 2 and 6, DNase I-cut DNA.

The overall results showed that sigma 54-DNA interactions at glnHp2 are changed by the action of activator in a nucleotide-dependent manner. Non-hydrolyzable nucleotide GTPgamma S did not substitute for dGTP to produce an extended footprint (data not shown). Qualitatively, the results are similar to those obtained with the S. meliloti nifH promoter (24) and clearly indicate that activator-dependent sigma 54 isomerization may be readily demonstrated with a number of different promoters.

Activator-dependent DNA Melting by sigma 54-- We used KMnO4 to probe for DNA melting within isomerized complexes. KMnO4 footprints at 30 °C using the S. meliloti nifH early melted DNA (-12/-11) in the isomerized complex did not convincingly show extra DNA melting, but the unpaired T residue at -12 of the heteroduplex region was much less reactive in both sigma 54-DNA complexes (data not shown). These KMnO4 footprints were repeated at 37 °C and with a promoter derivative having a single base pair of heteroduplex at -12 (top strand A replaced with C) (24). Results with the -12/-11 heteroduplex showed no extra DNA melting at the elevated temperature, but the unpaired T residue at -12 in the isomerized complex was more reactive to KMnO4 (data not shown). However, footprints using the C-12 heteroduplex DNA in the isomerized complex showed that, at 37 °C, the template strand T at -9 had a 2-fold increase in KMnO4 reactivity, indicating some extra DNA melting (data not shown). This contrasts with results obtained with glnHp2 derivatives (see below and Fig. 3), where considerable extra DNA melting in the isomerized complex was seen at 30 °C. It seems that melting of the nifH promoter within the isomerized complex occurs less frequently than with glnHp2 and may relate to differences in the ease with which the DNA strands of the two promoters can separate (see "Discussion").



View larger version (90K):
[in this window]
[in a new window]
 
Fig. 3.   DNA melting by sigma 54. Reactions were as described for Fig. 2B, except that KMnO4 replaced DNase I. Bound and unbound DNAs were isolated from the native gel, cleaved with piperidine, and analyzed as described for Fig. 2B. Sites of KMnO4 reactivity are numbered. Additions to each binding reaction are shown above each lane. Lanes 1, untreated DNA; lanes 2, KMnO4-treated DNA. A, template strand footprints; B, non-template strand footprints. RI, Region I.

Footprints of the glnHp2 -11/-10 promoter DNA show convincingly that the isomerized complex has extra DNA melting. As shown in Fig. 3A, sigma 54 strongly protected the template strand unpaired T residue at -11 from KMnO4 attack (compare lanes 2 and 3). In the isomerized complex, this protection was lost, and KMnO4 reactivity was evident at the unpaired T residue at -11 as well as at the new positions -9 and -7 (Fig. 3A, compare lanes 3 and 4). The bases at -11, -9, and -7 must be within an altered DNA structure compared with the free DNA and the non-isomerized sigma 54-DNA complex. It seems that activator brings about some extra DNA melting as well as changing the interaction of sigma 54 with the T residue at -11. The KMnO4 reactivity of the bands was quantified to enhance the reliability of the interpretation (Table I). The same patterns of enhanced reactivity and protection were seen in three independent experiments. Controls in which either activator or hydrolyzable NTP was omitted or a non-hydrolyzable NTP (GTPgamma S) was used showed that the extra DNA melting at -9 and -7 and the changed footprint at -11 required activator plus hydrolyzable nucleotide (data not shown). In the absence of these components, the sigma 54-DNA footprint remained unchanged, and the only complex evident in the gel shift assay was the fast running sigma 54-DNA complex (data not shown).


                              
View this table:
[in this window]
[in a new window]
 
Table I
Quantification of KMnO4 signals from Fig. 3
KMnO4 reactivities of T residues at positions -11, -9, and -7 (template strand) residues and -8 and -6 (non-template strand) in sigma -DNA complexes are expressed by their ratio to signals without protein (reactivity = 1; Fig. 3, A and B, lanes 2). S.D. values are in parentheses. sssigma 54-DNA, supershifted sigma 54-DNA complex; RI, Region I.

KMnO4 was used to probe the non-template strand in sigma 54-DNA and isomerized sigma 54-DNA complexes forming with the modified glnHp2 promoter. As shown in Fig. 3B (see also Table I), T residues at -8 and -6 were KMnO4-reactive in isomerized complexes that formed in response to activator and hydrolyzable nucleotide (Fig. 3B, lane 4). In the absence of activator and hydrolyzable nucleotide, the same sequences were no more reactive to KMnO4 than the naked DNA (Fig. 3B, compare lanes 2 and 3; see also Table I). As seen for the template strand (Fig. 3A), isomerization of the sigma 54-DNA complex was accompanied by some local DNA melting. In both cases, melting seems to extend from the -11/-10 heteroduplex region by at least (interpretation is limited by the placement of a potentially reactive T residue) an extra 4 base pairs toward the transcription start site (summarized in Fig. 7). Melting at these locations is seen in natural open promoter complexes forming with the sigma 54 holoenzyme (13-15, 29). The lack of reactivity of non-template T at -4 suggests either that the transcription start site sequence is not stably opened in the isomerized complex or that T at -4 is protected by sigma 54 from KMnO4 attack.

Weak Deregulated DNA Melting by sigma 54 Lacking Region I-- The amino-terminal 50 amino acids of sigma 54 (Region I) function to inhibit isomerization of the RNA polymerase holoenzyme as well as to promote enhancer responsiveness (30-33). Activities of Region I include contributions to the DNA-binding function of sigma 54, particularly the recognition and creation of the nucleated DNA near -12, and an interaction with core RNA polymerase (4, 16, 23, 31, 34). Removal of Region I results in activator-independent transcription if the DNA is transiently opened and allows the holoenzyme to engage with pre-melted DNA (11, 18, 20, 25). Using KMnO4 to probe the template strand of the glnHp2-Delta Isigma 54 complex, we found some evidence for weak activator-independent melting. As shown in Fig. 3A (lane 8), the T residue at -9 showed some increased KMnO4 reactivity in the Delta Isigma 54 complex compared with the zero protein control (lane 2). This region of extra DNA melting appears to be a subset of that found within the activator-dependent complex forming with full-length sigma 54 (Fig. 3A, compare lanes 4 and 8). The restricted pattern of melting at -9 seen with Delta Isigma 54 was independent of nucleotide and activator (Fig. 3A, compare lanes 7 and 8). Quantitative treatment of the KMnO4 reactivity (Table I) showed a constant increase in the reactivity of T at -9 when Delta Isigma 54 was bound. The increase was seen in three independent experiments. The T reside at -11 in the Delta Isigma 54 complex was slightly more protected compared with the isomerized sigma 54 complex (Fig. 3A), but significantly more reactive than sigma 54 alone to KMnO4 attack (compare lanes 3, 4, and 8; also see Table I). It seems that removal of Region I partially deregulates sigma 54 melting activity. Region I supplied in trans did not result in a significant change in KMnO4 reactivity to suggest a shift in the footprint toward that of the non-isomerized sigma 54-DNA complex (Fig. 3A, compare lanes 3 and 5; and Table I). It seems that, although Region I binds to the -12/-11-Delta Isigma 54 complex, this does not lead to large changes in KMnO4 reactivity (24).

KMnO4 was used to probe the non-template strand in the Delta Isigma 54-DNA complexes forming with the modified glnHp2 promoter. As shown in Fig. 3B (lanes 5-8) and Table I, no sequences were significantly more KMnO4-reactive than in the unbound DNA (Fig. 3B, lane 2). For the Delta Isigma 54-DNA complex, no further changes in KMnO4 reactivity were detected in response to activator and hydrolyzable nucleotide (Fig. 3B, lane 7). Region I supplied in trans to Delta Isigma 54-DNA binding assays did not change non-template strand KMnO4 reactivity (Fig. 3B, lanes 5 and 6).

Interactions of sigma 54 with DNA Pre-opened from -11 to -6 and a Gapped Structure-- The extra DNA opening from -9 to -6 seen in the activator-dependent isomerized complex (Fig. 3) could arise from activator functioning as a DNA helicase. To explore this issue, we wished to learn if pre-opening the DNA from -11 to -6 would allow the sigma 54 to bind promoter DNA in an isomerized state without activator. The interaction of sigma 54 with glnHp2 promoter DNA mismatched from -11 to -6 (see Fig. 1) to mimic the DNA opening seen in the activator-dependent isomerized complexes was examined in the presence and absence of activator. Gel shift assays showed that sigma 54 bound the -11/-6 opened DNA to give an initial complex (sssigma *-DNA) with reduced mobility compared with the -11/-10 opened DNA complex (Fig. 4, compare lanes 2 and 5). The reduced mobility was similar to that of the activator-dependent isomerized complex (sssigma -DNA) forming on the -11/-10 opened DNA (Fig. 4, compare lanes 3 and 5). The mobility of the sigma 54 complex with DNA opened from -11 to -6 was unchanged by activator and hydrolyzable nucleotide (Fig. 4, compare lanes 4 and 5). To more fully understand the properties of these slow running complexes, we probed them by DNase I and KMnO4 footprinting. Using glnHp2 promoter DNA mismatched from -11 to -6, sigma 54 gave a short DNase I footprint to -5 and no extra KMnO4 reactivity, and footprints were insensitive to activator and nucleotide (data not shown). We conclude that the reduced mobility of the sigma 54 complex with DNA opened from -11 to -6 is due to the altered DNA conformation and that pre-opening the DNA does not drive the change in sigma 54 needed for the extended downstream DNase I footprint to at least +2 seen in activator-dependent isomerized complexes (Fig. 2B, lane 4) (24). Instead, a change in sigma 54 conformation driven by the activator seems to be necessary for the extended footprint to +2. The insensitivity of the sigma 54 complex on the -11/-6 opened DNA to activator suggests that the DNA from -9 to -6 should be in a double-stranded form for activator to act on sigma 54, consistent with prior work with the S. meliloti nifH promoter (24).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4.   sigma 54-DNA complexes with glnHp2 -11/-6 and -11/-10 promoter DNAs have different gel mobilities. Shown are the results from the gel shift binding assay of sigma 54 with glnHp2 DNA (16 nM) opened from -11 to -6 compared with the -11/-10 opening. With glnHp2 -11/-6 DNA, a sigma 54-DNA complex (sssigma *-DNA) was formed that has mobility similar to that of the activator- and nucleotide-dependent glnHp2 -11/-10 supershifted complex (sssigma -DNA), but reduced mobility compared with the sigma -DNA complex. + indicates the presence of sigma 54 (1 µM), PspFDelta HTH (4 µM), and dGTP (4 mM). Heparin was added prior to gel loading. Results without heparin were similar.

We previously observed with the S. meliloti nifH promoter that removal of the top strand A-12 residue resulted in a sigma 54-DNA complex that did not form a new slow running species when incubated with activator and hydrolyzable or non-hydrolyzable nucleoside triphosphate (Fig. 5A, compare lanes 2, 4, and 5 with lane 6) (24). Delta Isigma 54 formed a slower running complex when Region I was in trans (Fig. 5A, compare lanes 2, 6, and 7). To characterize these complexes and to determine their relationship to the isomerized complex, we used DNase I and KMnO4 footprinting. The S. meliloti nifH promoter -12 gap (top strand) probe (see Fig. 1) gave a DNase I footprint with a distinct region that was cut poorly compared with the intact probe (data not shown). This region centered over the -12 gap and extended ~4 bases on either side, suggesting a locally altered DNA structure refractory to DNase I cutting. When sigma 54 bound, extra cutting from -10 to -1 was also evident, suggesting that sigma 54 stabilized a double-stranded DNA structure otherwise absent from the unbound gapped DNA (data not shown). There was no difference in the DNase I footprint under activating conditions (data not shown). The effects of activating conditions were then gauged by KMnO4 footprinting. When sigma 54 bound the gapped duplex, the single-stranded T residue at -12 (template strand) was protected from attack by KMnO4, and a modestly increased reactivity to KMnO4 was seen at T-9 indicative of some activator- and nucleotide-independent DNA melting (Fig. 5B, compare lanes 2 and 3). The same pattern was seen in the presence of activator and nucleotide (Fig. 5B, compare lane 3 with lanes 5-7) without evidence for extra activation-dependent melting. This suggests that a mismatch at -12 is needed for activator response. The slow running Delta Isigma 54 complex with Region I in trans footprinted like wild-type sigma 54 (Fig. 5B, compare lanes 3 and 8), whereas the Delta Isigma 54 complex showed less KMnO4 reactivity at T-9 (compare lanes 8 and 9). This suggests that Region I stabilizes the melted DNA at -9. The DNA melting seen with the gapped DNA when bound by sigma 54 (Fig. 5B) is consistent with the proposed role of the -12 nucleotide in restricting melting prior to activation (4). The gapped DNA allows melting within the sigma 54-DNA complex that is not evident with homoduplex DNA (Fig. 5B and data not shown).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Interaction of sigma 54 with -12 gapped DNA. A, gel mobility shift assay. Assays were conducted with sigma 54 or Delta Isigma 54 (1 µM), Region I (RI; 0.5 µM), end-labeled glnHp2 -12 gapped duplex (100 nM), PspFDelta HTH (4 µM), and dGTP or GTPgamma S (4 mM) where indicated (+). B, KMnO4 footprints. Assays were conducted as described for A and then treated with KMnO4. Template strand footprints are shown, and sites of KMnO4 reactivity are numbered. Additions to each binding reaction are shown above each lane. Lane 1, untreated DNA; lane 2, KMnO4-treated DNA.

Interactions of the sigma 54 Holoenzyme with Early Melted DNA-- The early melted DNA structure just downstream of the GC promoter that enables sigma 54 isomerization is believed to exist in closed promoter complexes, but is apparently absent in the activator-dependent open complex (23, 24). The chemical reactivities at -12/-11 seen in closed complexes are not evident in open complexes; rather, new melting is evident nearer the transcription start site (23). To explore the activator responsiveness of the sigma 54 holoenzyme on the S. meliloti nifH and E. coli glnHp2 early melted DNAs, we conducted gel shift and footprinting assays. As noted above, the sigma 54 holoenzyme bound to the early melted DNA assumes a complex with greater resistance to heparin than does the holoenzyme complex on homoduplex DNA (31). This has been suggested to involve a changed interaction between sigma 54 and core RNA polymerase since the binding of sigma 54 to core RNA polymerase in the absence of early melted DNA is heparin-sensitive (31, 34). Gel shift assays with either the S. meliloti nifH (Fig. 6A) or the E. coli glnHp2 (data not shown) early melted DNA in the presence of the sigma 54 holoenzyme showed, that under activating conditions, no new slow running holoenzyme-DNA complex was detected (compare lanes 6 and 7). A reduction in sigma 54 holoenzyme concentration led to increased formation of the supershifted sigma 54-DNA complex (sssigma -DNA, Fig. 6A, compare lanes 2-6) in the presence of activator and nucleotide, reflecting free sigma 54.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Holoenzyme binding to S. meliloti nifH early melted DNA. A, gel mobility shift assay. Reactions contained S. meliloti nifH -12/-11 DNA (16 nM). Holoenzyme (Esigma 54) was formed from 200 nM sigma 54 plus increasing amounts of core RNA polymerase (E) at 10, 25, 50, or 100 nM (indicated by the triangle above lanes 3-6). dGTP (1 mM), PspFDelta HTH (4 µM), and sigma 54 (200 nM) (lanes 1 and 2) were added where indicated (+). Unactivated holoenzyme (100 nM core RNA polymerase and 200 nM sigma 54)-DNA complex (Esigma -DNA) is shown in lane 7. After binding, heparin (100 µg/ml) was added for 5 min. No new sigma 54 holoenzyme-DNA species was detected under activation conditions. B, core binding to the sigma 54-DNA complex (graphed in C). A -35 to +6 base pair S. meliloti nifH -12/-11 DNA fragment (500 nM) (24) was used to form a supershifted complex (sssigma -DNA) with 300 nM sigma 54, 1 mM GTP, and 4 µM PspFDelta HTH (lane 2). GDP (10 mM) was added for 1 min to inhibit activator function, and then increasing amounts of core (E) were added (0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 µM) for 5 min before gel loading (lanes 3-8). Lane 9 shows core (1.0 µM) binding to DNA. C, quantification of complexes from B (lanes 2-8). , sigma 54 holoenzyme-DNA; , supershifted sigma 54-DNA complex; black-square, sigma 54-DNA.

We next used DNase I and KMnO4 footprinting to characterize the sigma 54 holoenzyme-DNA complexes on the early melted DNA and to learn if they had isomerized. Results showed that, under non-activating conditions, the isolated sigma 54 holoenzyme complex gave a short DNase I footprint to -5 and no extra KMnO4 reactivity (data not shown). The footprints were essentially as for sigma 54, except that some extra protection from DNase I by the sigma 54 holoenzyme upstream of -34 was observed with the E. coli glnHp2 promoter (data not shown). We next footprinted the sigma 54 holoenzyme-DNA complexes under activating conditions. The isolated holoenzyme complexes gave footprints indistinguishable from those obtained under non-activating conditions, suggesting that core-bound sigma 54 was not able to isomerize efficiently (data not shown). Further experiments with the use of initiating nucleotide (GTP) to potentially stabilize complexes on opened DNA through allowing initiation or with the omission of the heparin challenge to help preserve unstable complexes failed to produce sigma 54 holoenzyme footprints in which activator-dependent changes were evident (data not shown). We conclude that sigma 54 does not isomerize efficiently when bound to core RNA polymerase in assays using early melted DNA probes.

We confirmed (data not shown), using S. meliloti nifH -60 to +28 homoduplex DNA, that the heparin-resistant open promoter complexes formed by the action of activator and GTP had extended DNase I footprints to at least +13, definition of an exact end point being limited by the resolution of the gel and fragment size to +28 (14, 20, 35). However, the efficiency of activator-dependent stable complex formation was low in this assay. To address the issue that the sigma 54 holoenzyme bound to the early melted DNA might form new activator-dependent complexes (not distinguished from the activator-independent complexes because of the common property of heparin resistance) but with low efficiency, we also used KMnO4 as a probe of isomerization events. Here isomerization would be evident as increased reactivity to KMnO4 rather than protection from DNase I (see Fig. 3A, lane 4). Unlike the activator- and nucleotide-dependent complexes forming with sigma 54 and the early melted glnHp2 promoter DNA (Fig. 3A), the sigma 54 holoenzyme did not respond to activator to yield detectable extra melting or any associated loss of KMnO4 reactivity of the glnHp2 heteroduplex sequence (data not shown). The overall results show that the holoenzyme complex, in contrast to sigma 54, is poorly (if at all) responsive to activation conditions when bound to the early melted DNA. As discussed below, this may relate to unusually stable complex formation between the early melted DNA and the sigma 54 holoenzyme.

Core RNA Polymerase Binding to Isomerized sigma 54-DNA-- Having shown that sigma 54 bound to early melted DNA forms an isomerized complex (Ref. 24 and this work), but that sigma 54 holoenzyme does so inefficiently if at all (see above), we conducted an experiment to determine whether the conformation of the isomerized sigma 54-DNA complex allowed core RNA polymerase binding. In this assay, the isomerized complex was formed using an end-labeled -35 to +6 S. meliloti nifH -12/-11 DNA fragment (24) in excess of sigma 54 to diminish the amount of free sigma 54 and to ensure that core RNA polymerase interactions were potentially largely with DNA-bound sigma 54. Isomerization reactions were carried out and stopped by adding GDP (Fig. 6B, lane 2) (24). Increasing amounts of core RNA polymerase (E) were then added to bind sigma 54-DNA complexes. As shown in Fig. 6B, addition of increasing amounts of core RNA polymerase (lanes 3-8) depleted the sigma 54-DNA complex; and in parallel, an increasing amount of the sigma 54 holoenzyme bound to DNA (Esigma -DNA) was detected. The amount of isomerized sigma 54-DNA complex (sssigma -DNA) remained relatively constant throughout the titration with core RNA polymerase. It seems that core RNA polymerase preferentially binds the non-isomerized sigma 54-DNA complex. At high core RNA polymerase concentrations (in excess of 0.6 µM) (Fig. 6B, lanes 7 and 8), some core bound to DNA was detected, and this contributed to the apparent increased amount of sigma 54 holoenzyme bound (graphed in Fig. 6C) since core-DNA and holoenzyme-DNA complexes were not fully resolved. We infer that weak binding of the isomerized sigma 54-DNA complex by core RNA polymerase is because the interface between core and sigma 54 and DNA has changed upon isomerization. This leads to the suggestion that movements in sigma 54 and DNA are concerted with some in core RNA polymerase for forming the natural open promoter complex and is discussed below.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Melting by sigma 54-- Activator-dependent isomerization of sigma 54 results in the spreading of DNA melting away from the nucleated DNA structure and toward the transcription start site (Fig. 7). A structure melted over at least 6 base pairs is generated. Interactions with the nucleated DNA located at -11/-10 are lessened in the isomerized complex compared with the initial sigma 54-DNA complex. Although removal of Region I sequences allows some weak DNA melting by sigma , this is not as extensive as activator-driven isomerization. A comparison of results obtained with Delta Isigma 54 and intact sigma 54 shows that the full DNA-melting activity of sigma 54 has some determinants outside of regulatory Region I (for weak melting at -9) and some that depend upon Region I (for extra melting from -8 to -6).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Summary of footprint results. The extents of DNase I protection, sites of KMnO4 reactivity, and protection are shown. ------, DNase I protection; full () and partial () protection from KMnO4 attack, respectively; * and , enhanced reactivity in complexes forming with sigma 54 or its Region I-deleted variant (Delta Isigma 54) or in activator-dependent sigma 54 isomerized complexes (sssigma 54).

Isomerized sigma 54-nifH promoter complexes failed to show KMnO4 reactivity as extensively as glnHp2 (Fig. 3 and data not shown). If this reflects less melting (as opposed to shielding from KMnO4), differences in intrinsic DNA opening rates between the two promoters in combination with the sequence-independent single-stranded DNA-binding activity in sigma 54 (36) could contribute. DNA opening at -9 seen in natural nifH open complexes but weakly detected in isomerized complexes with sigma 54 might therefore reflect a stabilizing contribution from the core enzyme (37). Clearly, changes in the sigma 54-DNA relationship seen in DNase I footprints of isomerized complexes in which melting may not have occurred is consistent with the idea that activator changes sigma 54 structure and that pre-opening the DNA does not drive this change (22, 24).

Comparisons of the closed and activator-dependent open sigma 54 holoenzyme-promoter complexes using KMnO4 and ortho-copper phenanthroline footprinting suggest that the structure of the DNA immediately downstream of the GC element differs between these complexes and that contact with G-13 is also altered (14, 15, 23). These observations are fully consistent with the lessened sigma  interaction at -11 detected by the KMnO4 footprints of isomerized complexes reported here (Fig. 3). Based on the known interaction between Region I and core polymerase (34, 38), we suggested that the silencing interactions associated with the -13 to -11 sigma 54-DNA contact (4) can contribute significantly to inhibiting RNA polymerase isomerization through restricting conformational changes within the core subunits (16). Changes in promoter DNA and sigma 54 holoenzyme conformation are probably coupled through sigma 54 Region I to maintain either the closed or open state of the promoter. Open promoter complexes that form in deregulated transcription by the sigma 54 holoenzyme are unstable compared with activator-dependent open complexes (18, 19, 25). Their instability may be related to incomplete DNA melting beyond -9 as a consequence of the mutations in Region I of sigma 54 and a need for Region I to stabilize melted DNA (Fig. 5B).

Role of Activator-- Results with DNA mismatched from -11 to -6 across the sequences melted in the isomerized sigma 54-DNA complex support the view that activator drives a conformational change in sigma 54 rather than generating isomerization by solely creating an opened DNA structure for sigma 54 binding. For both transcription and sigma 54 isomerization, pre-opening of the DNA through heteroduplex formation does not by pass activator requirements (22), unless the structure recognized by sigma 54 at -11 is destroyed (36). Rather, activator-dependent conformational changes in sigma 54 and the holoenzyme seem necessary for open complex formation and sigma 54 isomerization (24, 36). DNase I footprinting shows that sigma 54 clearly binds to double-stranded DNA ahead of the locally melted DNA or the fork junction that forms next to the GC promoter element in closed complexes (this work and Refs. 4 and 24). This suggests that the further melting of the double-stranded DNA within the sigma 54-DNA complex is an active process in the sense that it is not a primary result of a domain of sigma 54 translocating along the duplex and trapping DNA strands at a fraying fork junction. Energy for duplex destabilization may come from the tight binding of sigma 54 to the initially locally melted DNA. An activator-driven change in sigma 54-DNA interaction might release sigma 54 from binding to the double-stranded DNA downstream of the -12 GC and switch sigma 54 to a conformation that then allows it to bind single-stranded DNA. Combined with the single-stranded DNA-binding activities in sigma 54 (4, 36) and the core RNA polymerase (39), sigma 54 isomerization would lead to formation of the stable open promoter complex.

Stable Holoenzyme Binding-- When bound to core RNA polymerase, activator-dependent isomerization of sigma 54 was not evident, in marked contrast to its efficient isomerization without core. It seems that the short sequence of heteroduplex downstream of GC inhibits isomerization of sigma 54 within the holoenzyme since, on linear DNA, activator-dependent isomerization of the holoenzyme is evident (11, 23, 39). The activator-dependent movement of sigma 54 across the sequence opened downstream of GC implied by the results of our KMnO4 footprints may be inhibited when sigma 54 is bound by core RNA polymerase. This suggests that isomerization of sigma 54 within the holoenzyme may normally require that the local DNA opening next to GC does not strongly persist. In the homoduplex, the DNA can base pair again, but not in the heteroduplex. Consistent with this view is the observation that the local DNA distortions downstream of GC and present in the closed complex are apparently changed in the open complex, as judged by the reduced sensitivity of the DNA to two chemical probes of DNA structure (23). The spread of melting observed in the sigma  isomerization assays would then normally be associated with a changing of the DNA structure believed to be locally melted in the closed complex, achieved through a breaking of DNA contacts and a rebinding of sigma 54 to DNA. These considerations suggest an ordered series of events in which changes in core RNA polymerase to sigma 54 interactions and sigma 54-DNA interactions occur in response to activator to allow sigma 54 isomerization and the holoenzyme to progress from the closed complex to the open complex. This view is consistent with activator interacting with both core RNA polymerase and sigma 54 (24, 40) to achieve isomerization of the holoenzyme and with the view that the promoter sequences around GC contribute to preventing the holoenzyme from isomerizing prior to activation and to set the target of the activator (4, 24).

Core-sigma Interactions and DNA Melting-- Results of core RNA polymerase binding with the isomerized and non-isomerized sigma 54-DNA complexes strongly suggest that some points of interaction between sigma 54 and core are changed upon isomerization of the sigma 54-DNA complex. The poorer core binding of the isomerized complex likely correlates with the changes in protease sensitivity of sigma 54 in the isomerized complex (24). Changed DNA structure within the isomerized sigma 54-DNA complex may also contribute to poor binding by core RNA polymerase. The strong reduction in isomerization of sigma 54 resulting from core RNA polymerase binding prior to exposure to activating conditions (Fig. 6A) and the weak binding of isomerized sigma 54 to core RNA polymerase (Fig. 6, B and C) is striking. It seems that normally for efficient sigma 54 holoenzyme isomerization and open complex formation, activator-dependent changes in sigma 54 structure would occur in concert with a changed binding of parts of sigma 54 to core RNA polymerase; but on the early melted DNA, this is not occurring properly. As indicated above, when using the early melted DNA as template, the failure to reconfigure interactions with DNA next to the GC promoter may simply strongly stabilize a sigma 54 conformation that is unfavorable for core RNA polymerase binding. We therefore suggest that, in closed complexes, activator drives a conformational change in sigma 54 that results in altered contacts with the early melted DNA (as detected in our KMnO4 footprinting; see above), allowing binding of sigma 54 and core RNA polymerase to permit full sigma 54 isomerization and the associated isomerization of the closed complex to the open complex. It seems that Region I of sigma 54 greatly contributes to these events through (i) its requirement for creating the early melted DNA when the holoenzyme binds homoduplex DNA, (ii) directing sigma 54 binding to the early melted DNA in heteroduplexes and associated fork junction structures, (iii) a binding interaction with core RNA polymerase, and (iv) the changing Region I structure in isomerized sigma 54 and the activated sigma 54 holoenzyme (reviewed in Ref. 12). The recent demonstration that Region I sequences localize over the -12 promoter region is fully consistent with these observations and points to a central role of the protein and DNA elements that localize there in establishing new interactions that allow DNA melting and a changing binding relationship between core subunits and sigma 54 (41). We also note that the refractory behavior of the sigma 54 holoenzyme bound to the early melted DNA and the poor core binding of isomerized sigma 54 are fully consistent with the view that interactions sigma 54 makes with the -12 promoter region, in particular sequences just downstream of GC, are key in limiting spontaneous activator-independent open complex formation (4).


    ACKNOWLEDGEMENTS

We thank Jörg Schumacher and Susan Jones for useful comments on this manuscript.


    FOOTNOTES

* This work was supported by a Wellcome Trust grant (to M. B.) and by a Biotechnology Marie Curie fellowship (to M.-T. G.). Work was conducted in the Imperial College Center for Structural Biology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biology, Sir Alexander Fleming Bldg., Imperial College of Science Technology and Medicine, London SW7 2AZ, UK. Tel.: 207-594-5442; Fax: 207-594-5419; E-mail: m.buck@ic.ac.uk.

Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007779200


    ABBREVIATIONS

The abbreviation used is: GTPgamma S, guanosine 5'-O-(3- thiotriphosphate).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Gross, C. A., Chan, C., Dombroski, A., Gruber, T., Sharp, M., Tupy, J., and Young, B. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 141-155[Medline] [Order article via Infotrieve]
2. Fenton, M. S., Lee, S. J., and Gralla, J. D. (2000) EMBO J. 19, 1130-1137[Abstract/Free Full Text]
3. Guo, Y., and Gralla, J. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11655-11660[Abstract/Free Full Text]
4. Guo, Y., Wang, L., and Gralla, J. D. (1999) EMBO J. 18, 3736-3745[Abstract/Free Full Text]
5. Helmann, J. D., and deHaseth, P. L. (1999) Biochemistry 38, 5959-5967[CrossRef][Medline] [Order article via Infotrieve]
6. Malhotra, A., Severinova, E., and Darst, S. A. (1996) Cell 87, 127-136[Medline] [Order article via Infotrieve]
7. Marr, M. T., and Roberts, J. W. (1997) Science 276, 1258-1260[Abstract/Free Full Text]
8. Callaci, S., and Heyduk, T. (1998) Biochemistry 37, 3312-3320[CrossRef][Medline] [Order article via Infotrieve]
9. Callaci, S., Heyduk, E., and Heyduk, T. (1999) Mol. Cell 3, 229-238[Medline] [Order article via Infotrieve]
10. Cannon, W. V., Chaney, M. K., Wang, X.-Y., and Buck, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5006-5011[Abstract/Free Full Text]
11. Cannon, W., Gallegos, M.-T., Casaz, P., and Buck, M. (1999) Genes Dev. 13, 357-370[Abstract/Free Full Text]
12. Buck, M., Gallegos, M.-T., Studholme, D. J., Guo, Y., and Gralla, J. D. (2000) J. Bacteriol. 182, 4129-4136[Free Full Text]
13. Morett, E., and Buck, M. (1989) J. Mol. Biol. 210, 65-77[Medline] [Order article via Infotrieve]
14. Popham, D. L., Szeto, D., Keener, J., and Kustu, S. (1989) Science 243, 629-635[Medline] [Order article via Infotrieve]
15. Sasse-Dwight, S., and Gralla, J. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8934-8938[Abstract]
16. Chaney, M., and Buck, M. (1999) Mol. Microbiol. 33, 1200-1209[CrossRef][Medline] [Order article via Infotrieve]
17. Wang, J. T., Syed, A., Hsieh, M., and Gralla, J. D. (1995) Science 270, 992-994[Abstract]
18. Wang, J. T., Syed, A., and Gralla, J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9538-9543[Abstract/Free Full Text]
19. Wang, L., Guo, Y., and Gralla, J. D. (1999) J. Bacteriol. 181, 7558-7565[Abstract/Free Full Text]
20. Wang, J. T., and Gralla, J. D. (1996) J. Biol. Chem. 271, 32707-32713[Abstract/Free Full Text]
21. Wang, L., and Gralla, J. D. (1998) J. Bacteriol. 180, 5626-5631[Abstract/Free Full Text]
22. Wedel, A., and Kustu, S. (1995) Genes Dev. 9, 2042-2052[Abstract]
23. Morris, L., Cannon, W., Claverie-Martín, F., Austin, S., and Buck, M. (1994) J. Biol. Chem. 269, 11563-11571[Abstract/Free Full Text]
24. Cannon, W., Gallegos, M.-T., and Buck, M. (2000) Nat. Struct. Biol. 7, 594-601[CrossRef][Medline] [Order article via Infotrieve]
25. Gallegos, M.-T., Cannon, W., and Buck, M. (1999) J. Biol. Chem. 274, 25285-25290[Abstract/Free Full Text]
26. Jovanovic, G., Rakonjac, J., and Model, P. (1999) J. Mol. Biol. 285, 469-483[CrossRef][Medline] [Order article via Infotrieve]
27. Claverie-Martín, F., and Magasanik, B. (1992) J. Mol. Biol. 227, 996-1008[Medline] [Order article via Infotrieve]
28. Sasse-Dwight, S., and Gralla, J. D. (1989) J. Biol. Chem. 264, 8074-8081[Abstract/Free Full Text]
29. Claverie-Martín, F., and Magasanik, B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1631-1635[Abstract]
30. Casaz, P., Gallegos, M.-T., and Buck, M. (1999) J. Mol. Biol. 292, 229-239[CrossRef][Medline] [Order article via Infotrieve]
31. Gallegos, M.-T., and Buck, M. (2000) J. Mol. Biol. 297, 849-859[CrossRef][Medline] [Order article via Infotrieve]
32. Syed, A., and Gralla, J. D. (1997) Mol. Microbiol. 23, 987-995[Medline] [Order article via Infotrieve]
33. Syed, A., and Gralla, J. D. (1998) J. Bacteriol. 180, 5619-5625[Abstract/Free Full Text]
34. Gallegos, M.-T., and Buck, M. (1999) J. Mol. Biol. 288, 539-553[CrossRef][Medline] [Order article via Infotrieve]
35. Tintut, Y., Wang, J. T., and Gralla, J. D. (1995) Genes Dev. 9, 2305-2313[Abstract]
36. Guo, Y., Lew, C. M., and Gralla, J. D. (2000) Genes Dev. 14, 2242-2255[Abstract/Free Full Text]
37. Cannon, W., Missailidis, S., Smith, C., Cottier, A., Austin, S., Moore, M., and Buck, M. (1995) J. Mol. Biol. 248, 781-803[CrossRef][Medline] [Order article via Infotrieve]
38. Casaz, P., and Buck, M. (1999) J. Mol. Biol. 285, 507-514[CrossRef][Medline] [Order article via Infotrieve]
39. Brodolin, K., Mustaev, A., Severinov, K., and Nikiforov, V. (2000) J. Biol. Chem. 275, 3661-3666[Abstract/Free Full Text]
40. Lee, J. H., and Hoover, T. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9702-9706[Abstract]
41. Wigneshweraraj, S. R., Fujita, N., Ishihama, A., and Buck, M. (2000) EMBO J. 19, 3038-3048[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.