Conserved Regions 4.1 and 4.2 of (cid:1) 70 Constitute the Recognition Sites for the Anti- (cid:1) Factor AsiA, and AsiA Is a Dimer Free in Solution*

The association of the bacteriophage T4-encoded AsiA protein with the (cid:1) 70 subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and (cid:1) 70 have been elucidated. Residues throughout the N-termi-nal half of AsiA are involved either directly or indirectly in binding to (cid:1) 70 whereas the two highly conserved C-terminal regions of (cid:1) 70 , denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of (cid:1) 70 . Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1

The integrity of bacterial transcription initiation relies on specificity in promoter recognition by the RNA polymerase holoenzyme. This specificity is conferred upon the holoenzyme by the subunit (or factor). The subunit associates reversibly with the polymerase core subunits (␣ 2 ␤␤'), and it orchestrates transcription initiation through interactions with specific promoter sequences. In most bacteria, a primary factor, which is essential for survival, is responsible for the bulk of transcription initiation, including transcription of the housekeeping genes. Promoter sequences not recognized by the pri-mary , however, require alternate factors that are produced or mobilized in response to specific stimuli. These alternate factors permit both diversity and specificity in promoter recognition by the RNA polymerase holoenzyme. In general, factors also represent important and useful control elements for regulating transcription initiation and gene expression.
One avenue for regulation of transcription initiation involves post-translational inhibition of function by interactions of factors with anti-factors. Anti-factors modify the activity of the RNA polymerase holoenzyme through interactions with specific primary or alternate factors. Many specific /antipairs have been identified, and their biological roles have been initially characterized and summarized (1)(2)(3)(4). Only recently have some of the structural features of antifactors, the interfaces they share with their cognate factors, and structural details of /antiinteractions begun to be elucidated (5)(6)(7)(8)(9).
The bacteriophage T4-encoded AsiA protein, the first antifactor to be discovered, binds tightly to the 70 subunit of the Escherichia coli RNA polymerase holoenzyme (10 and references therein). AsiA is a small (90 amino acids), stable globular protein, composed exclusively of helix and coil regions (7). AsiA has no known sequence homologs. AsiA is intimately involved in both inhibition of transcription at bacterial and early phage promoters and in facilitating transcription at phage middle promoters (11)(12)(13)(14)(15)(16). Thus, a detailed analysis of the interaction between AsiA and 70 is crucial for understanding transcription regulation during T4 infection and for a comprehensive appreciation of function in general.
The AsiA binding determinants of 70 have been localized to the C-terminal domain (15,(17)(18)(19)(20). The C-terminal 108 amino acids of 70 ( 506 70 , Fig. 1) binds to AsiA with an affinity equaling (15) or exceeding (20) that of full-length 70 , indicating that all of the AsiA binding determinants of 70 reside within 506 70 . Thus, in terms of AsiA binding, 506 70 is an excellent functional analogue of full-length 70 , and the AsiA-506 70 complex can serve as a suitable model of the native interaction.
The C-terminal domain of 70 harbors several highly conserved regions (21,22). Of these, only region 4.2 ( Fig. 1) has heretofore been shown to interact with AsiA (17)(18)(19). In the RNA polymerase holoenzyme, region 4.2, a putative helix-turnhelix motif (21,23), interacts with specific DNA sequences in the Ϫ35 promoter consensus element (21,24,25), providing promoter recognition specificity. However, free 70 does not bind DNA. DNA binding by free 70 is precluded by an intramolecular interaction involving regions 1.1 and 4 that presumably occludes region 4.2 (24,25). Conversely, AsiA binds to both free 70 and 70 in the RNA polymerase holoenzyme. This difference might simply reflect the relative affinities of AsiA and DNA for 4.2. Alternatively, ancillary AsiA binding sites on 70 could serve to increase the affinity of 70 for AsiA or provide an avenue for interaction not occluded intramolecularly by 70 . Prospective additional AsiA binding sites on 70 include conserved regions 4.1 and 3.2.
The role of conserved region 4.1 (Fig. 1) is not well understood. It is less highly conserved than 4.2 among primary factors. Like 4.2, its secondary structure is predicted to be predominantly helical (21). It has been suggested that it stabilizes the putative helix-turn-helix DNA binding motif of 4.2 (21). Region 4.1 of F (not a primary ) of Bacillus subtilis recognizes an anti-factor, SpoIIAB (26), demonstrating a pivotal regulatory role for 4.1 of F .
Our results define the oligomeric state of AsiA, delineate the AsiA binding determinants of 70 , and localize the 70 binding interface on AsiA. Additionally, our results expose a substantial reorganization of the AsiA structure, presumably involving the dimer interface, upon interaction with 70 . Finally, to characterize structurally the interaction of AsiA with 70 in solution (using NMR), minimal models of 70 are needed. The minimal 70 models should mimic the native condition, including high affinity AsiA binding and native-like perturbation of AsiA structure. Our results reveal functionally relevant minimal 70 models.

EXPERIMENTAL PROCEDURES
AsiA Production and Labeling-The cloning, expression, and overproduction of electrophoretically pure AsiA protein have been described (11)(12)(13). Uniformly 15 N-labeled AsiA was produced by growth on 15 Nlabeled Isogro (Isotec, Inc., Miamisburg, Ohio) media (7). Uniformly 13 C, 15 N-labeled AsiA was produced by growth on minimal media with uniformly 15 N-labeled NH 4 Cl and uniformly 13 C-labeled glucose as the sole nitrogen and carbon sources.
Analytical Ultracentrifugation-A Beckman Optima XL-I analytical ultracentrifuge with a four-position AN-Ti rotor was used for the sedimentation equilibrium experiment. The protein solution (0.1 mM 15 Nlabeled AsiA in 50 mM sodium acetate, pH 6.3) and the reference buffer were loaded into the right and left sector, respectively, of a doublesector 1.2-cm cell. The samples were equilibrated at 40,000 rpm and 4°C for ϳ48 h. To monitor the approach to equilibrium, the absorption profile in the cell was measured every 4 h. The data were analyzed assuming a single-component model using the software supplied with the instrument. The partial specific volume of AsiA (0.722 ml/g) was calculated as described previously (27). The density of the buffer was approximated by the density of water at 4°C.
Peptide Production-Peptides corresponding to conserved regions 3.2, 4.1, and 4.2 ( Fig. 1) of E. coli 70 were synthesized commercially (SynPep Corp., Dublin, CA) and purified using reversed-phase HPLC. 1 The peptide corresponding to the C-terminal 108 amino acids of E. coli 70 ( 506 70 ) was expressed as a GST fusion using a pGEX plasmid (gift from Carol Gross to E. N. Brody) in E. coli BL21. The fusion protein was solubilized from inclusion bodies using 6 M urea, with renaturation by dialysis and purification using standard GST protocols. After thrombin cleavage, only some of the free GST could be removed by HPLC. It has been shown (15) and corroborated (the results herein) that GST does not affect the binding of AsiA to 506 70 . Complexes of AsiA with 70 -derived Peptides-Complexes of 15 Nlabeled AsiA with 70 -derived peptides were prepared by incremental addition of the peptides in solution (50 mM sodium acetate-d 3 , 10% D 2 O, pH 6.2) to AsiA in the same solution. The final concentrations of the complexes in ϳ600 l were ϳ0.5 mM.
NMR Spectroscopy-NMR spectra were acquired with a Varian INOVA spectrometer operating at 600 MHz ( 1 H), at a sample temperature of 25°C. Gradient sensitivity-enhanced 1 H, 15 N-HSQC (28) spectra were recorded as detailed recently (7), with 1024 and 128 complex points in the 1 H (t2) and 15 N (t1) dimensions, respectively (spectral  13 C F 1 -filtered, F 3 -edited NOESY-HSQC spectra, permitting selective observation of NOEs between unlabeled and 13 C, 15 N-labeled protomers in an AsiA dimer, confirm the existence of the AsiA dimer. A, a small section from the methyl region of a 1 H, 13 C-HSQC spectrum of AsiA. B, the same region from a two-dimensional 13 C F 1 -filtered, F 3edited NOESY-HSQC spectrum of a solution containing 50% unlabeled and 50% 13 C, 15 N-labeled AsiA (ϳ1.5 mM AsiA). The observed peaks correspond to contacts (NOEs) between isotopically labeled and unlabeled protomers in the dimer. C, a section of a three-dimensional filtered/edited experiment (a two-dimensional, 1 H-1 H slice at 13 C ϭ 17.85 ppm) using the same sample as in B, revealing many NOEs between the AsiA protomers.

RESULTS AND DISCUSSION
The oligomeric state of AsiA was determined using analytical ultracentrifugation (Fig. 2). The results indicate that AsiA is a dimer. Because NMR spectra show a single set of resonances for the 90 amino acid residues, the dimer is symmetric (on the NMR chemical shift time scale), and the dissociation constant for the dimer is small (no monomer is observed). Preliminary results of fluorescence studies of AsiA mutants containing tryptophan and 7-azatryptophan (not shown) also indicate a very low dissociation constant for the dimer.
Confirmation of the dimeric structure of AsiA and exchange of subunits between AsiA dimers were addressed using NMR (Fig. 3). Using a sample composed of 50% uniformly isotopically ( 13 C, 15 N) labeled AsiA and 50% unlabeled AsiA, NOEs between unlabeled and labeled protomers are observed (Fig. 3, B and C, with a 1 H, 13 C-HSQC spectrum shown in Fig. 3A for reference). Control experiments using either all isotopically labeled or all unlabeled AsiA confirm that the interprotomer NOEs are genuine. In addition, a sample of AsiA (50% isotopically labeled, 50% unlabeled) that had been denatured with urea and subsequently renatured by dialysis showed interprotomer NOEs identical to those without the denaturation/renaturation step (not shown). A thorough examination of the residues at the dimer interface and the specific interprotomer contacts, in con-junction with determination of the high resolution solution structure of the AsiA dimer, is ongoing.
Conserved region 4.2 is not the only AsiA binding determinant in 70 (Fig. 4). The chemical shift changes in AsiA induced by binding to 506 70 are distinct from those induced by binding of 4.2 70 (Fig. 4, A and C). Because all of the AsiA binding regions of 70 are harbored by the 506 70 peptide (15,(17)(18)(19)(20), these results suggest that 4.2 is not the only AsiA binding site in 70 . As expected, the AsiA-506 70 complex is in slow exchange with its dissociated components on the NMR chemical shift time scale, confirming tight binding (15). Binding of 4.2 70 to AsiA is also tight, as this complex also is in slow exchange with its dissociated components. Although it is known that 4.2 binds to AsiA in solution (17)(18)(19), our results show that additional regions of 70 , within 506 70 , also interact with AsiA. Conserved region 4.l of 70 also binds to AsiA (Fig. 4B) binding. This is the first unambiguous evidence for binding of AsiA to residues in the 4.1 region of 70 .
The peptide corresponding to region 3.2 of 70 was also tested for binding to AsiA using NMR. No interaction was observed. These results, however, do not preclude a very weak interaction.
In addition to binding individually to AsiA, the 4 peptides also bind simultaneously to AsiA (Fig. 4D).  Fig. 4, B-D, and Fig. 5, A-C, reveals this is not the case, again indicating that 4.1 70 and 4.2 70 can indeed bind simultaneously to AsiA. Finally, binding of a single peptide to AsiA with binding of a second peptide to the first (without contacts between AsiA and the second peptide) is not precluded by the data.
NMR chemical shift changes of protein resonances accompanying ligand binding report on ligand-induced structural perturbations, and within limits (31), can define and localize intermolecular interfaces (for example, Refs. 32 and 33). The minimal chemical shift perturbation method (32) can be used to obtain an unambiguous but conservative estimate of chemical shift changes and to map binding interfaces. Unequivocally, the chemical shifts of many residues in the N-terminal half of AsiA, including each of the three helical regions, are perturbed significantly upon 506 70 binding (Fig. 6), suggesting that many residues in the N-terminal half of AsiA, including all three N-terminal helices (7), are involved either directly or indirectly in binding to 70 . From the results of a recent study using deletion mutants (8), it was concluded that the first 20 amino acids of AsiA are both necessary and sufficient to bind 70 and inhibit transcription. However, the results in Fig. 6 clearly suggest that secondary or tertiary structural changes occur in many regions of the N-terminal half of AsiA when 506 70 binds, and these are not limited to the first 20 residues.
The 70 -derived peptides must compete with an interaction within the AsiA dimer for a binding site on AsiA. This is signified by the fact that, for instance, even though the 4.1 70 peptide binds tightly to AsiA (slow exchange complex), a significant excess of (free) 4 AsiA-4.1 70 complex (not shown). Similar behavior is noted with the 4.2 70 peptide, although a somewhat smaller excess of peptide is necessary in this case. This behavior clearly indicates a competition for a binding site (and also precludes a simple conclusion as to the stoichiometry of the complexes). The equilibrium constant between the rearranged form of AsiA (which binds the 70 peptides) and the unbound form favors the unbound form; hence the competition. Although the nature of this event/ rearrangement of AsiA upon binding to the 70 -derived peptides is not known, a rearrangement of the dimer interface is suggested from preliminary assignments for the interprotomer NOEs in AsiA, which indicate that many residues whose chemical shifts change upon binding to the 70 peptides are also located at the dimer interface. Our current hypothesis, based on these results and symmetry arguments (below), is that the AsiA dimer dissociates upon binding to the 70 -derived peptides.
AsiA is a symmetric dimer, and the symmetry is apparently not lost upon binding to the 70 -derived peptides because the NMR spectra of the complexes show a single set of AsiA resonances. Binding of a single (asymmetric) peptide to an AsiA dimer would destroy the symmetry, resulting in observation of resonances in the NMR spectra for each of the two (now nonequivalent) protomers. Thus, either an even, integral number of a given 70 -derived peptide must bind in a symmetrical fashion to each AsiA dimer (most likely two), or the AsiA dimer must dissociate upon peptide binding. Consequently, a complex of one AsiA dimer with a single 506 70 peptide (or a single 4.1 70 or 4.2 70 peptide) would appear to be ruled out as an acceptable stoichiometry. Further experimentation to test for dissociation of AsiA upon peptide binding and to confirm the stoichiometry of the complexes is underway using analytical ultracentrifugation, gel shift assays combined with quantitative amino acid analysis, and 13 C F 1 -filtered, F 3 -edited NMR experiments of complexes of AsiA with 70 -derived peptides.
The existence of region 4.1 of 70 as an additional, or alternative, site for interaction with AsiA provides potential explanations for the ability of AsiA to bind to free 70 whereas DNA cannot (34) and permits models for AsiA function that are not dependent on a single AsiA binding site on 70 . In free 70 , intramolecular inhibition involving regions 1.1 and 4 obscures the two DNA binding elements, regions 4.2 and 2.4 (24,25). The affinity of DNA for region 4.2 is apparently smaller than is necessary to compete effectively with the autoinhibition. The additional affinity afforded AsiA by binding to both regions 4.1 and 4.2 may in itself permit effectual competition with the intramolecular, autoinhibitory interaction. However, depending on the degree to which region 4.1 is occluded intramolecularly, the high affinity of AsiA for 4.1 may prove entirely responsible for binding free 70 to AsiA, or a transient association of AsiA with 4.1 alone may prove to be a crucial initial intermediate in the recognition process. Finally, because AsiA serves a dual role, functioning both to inhibit transcription at early promoters and to promote transcription (along with MotA) at middle promoters, AsiA function may be dependent upon which site(s) on 70 it is interacting with. Discrete functional states might include AsiA interacting with only region 4.1, with only region 4.2, or with both 4.1 and 4.2 simultaneously.
These assertions are consistent with the results of Sharma et al. (20), which show that C-terminal fragments of 70 bind to AsiA with affinities exceeding that of free, intact 70 . This is due either to partial occlusion of both Our studies reveal the significance of both regions 4.1 and 4.2 of 70 in the interaction of 70 with AsiA. The complexes between AsiA and the 70 -derived peptides described in these studies will additionally provide the opportunity for high resolution characterization of the structural and functional coupling between conserved regions 4.1 and 4.2 of 70 that promote transcriptional regulation by AsiA. Our current efforts include determination of the high resolution solution structure of AsiA alone and complexed to the AsiA binding regions of 70 . Finally, our results suggest the utility of 70 -derived peptides as unique and valuable probes of bacterial transcription and perhaps of other /antiinteractions.