Journal of Bacteriology, July 2002, p . 3957-3964, Vol . 184, No . 14

The MotA protein binds as a monomer (6, 33) to a 9-bp element (MotA box) centered at position -30 of middle promoter DNA (3, 19, 23) . In addition, MotA forms a complex with ⁷⁰ (18) . Nuclear magnetic resonance and crystallographic studies indicate that the 211 amino acids of MotA are organized into an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a small flexible linker (15, 33, 34) . Mutations within the NTD of MotA eliminate transcriptional activation and the formation of the MotA-⁷⁰ complex (14, 18), suggesting that the NTD is the activation domain of MotA . Transcriptional activation of middle promoters also requires the T4 coactivator AsiA, which binds very tightly to ⁷⁰ (44, 55, 56) . Binding sites for AsiA have been mapped within C-terminal amino acids (regions 4.1 and 4.2) of ⁷⁰ (8, 49, 50, 53, 59) . Residues within region 4.2 normally contact the -35 element of host promoter DNA (5, 9, 17, 29, 54) . In the absence of MotA, AsiA binding to ⁷⁰ inhibits transcription by polymerase from promoters that require recognition of the -35 canonical sequences (8, 45, 51), suggesting that the presence of AsiA inhibits the ⁷⁰ region 4.2-DNA interaction .

In this paper we show that the interaction of a MotA N-terminal peptide (amino acids 1 to 97) with ⁷⁰, like the interaction of AsiA with ⁷⁰, involves the C-terminal region of ⁷⁰ . In addition, deletions of the amino acids within the far-C-terminal region of ⁷⁰ (amino acids 604 to 613) impair the ability of RNA polymerase to perform MotA-dependent activation in vitro . We also show that a MotA C-terminal peptide, beginning at amino acid 102, binds DNA with an apparent dissociation constant like that of wild-type MotA . Our results support a model for MotA-dependent activation in which the interaction between the DNA-bound MotA and the C-terminal region of ⁷⁰ helps to substitute functionally for an interaction between ⁷⁰ and a promoter -35 element .

DNA. Oligonucleotide primers were obtained from Gene Probe Technologies and Cruachem Inc . (Primer sequences are available upon request.) The 5'-³²P-labeled 74-bp P_uvsX DNA, containing the P_uvsX sequences from positions -56 to +18, was obtained as described previously (23) . pDKT90, which contains the T4 middle promoter P_uvsX, has been described elsewhere (37) . Linear templates for transcription, obtained by BsaAI restriction of pDKT90, were purified by phenol extraction followed by ethanol precipitation .

The pBR-⁷⁰ chimera plasmid (11) contains a ColE1 replication origin, confers carbenicillin resistance, and directs transcription of an -⁷⁰ chimera gene under the control of the tandem promoters P_lpp and P_lacUV5 . The resulting -⁷⁰ chimera protein is composed of the NTD of (amino acids 1 to 248) fused in frame to the C-terminal region of ⁷⁰ (amino acids 528 to 613) . The pBR-⁷⁰ derivative encoding -⁷⁰(R596H) has been described previously (11), and the pBR-⁷⁰ derivatives encoding -⁷⁰ chimeras bearing substitutions H600A, H600R, and R608C were constructed similarly . All of these derivatives are identical to pBR-⁷⁰ except for the indicated changes . The -³⁸ chimera plasmid, pBR-³⁸ (11), is identical to pBR-⁷⁰ except that all of the ⁷⁰ sequences have been replaced with the comparable ³⁸ sequences (encoding amino acids 243 to 330) . The ⁷⁰/³⁸ hybrid plasmid, pBR-⁷⁰/³⁸, was constructed by replacing the ⁷⁰ sequences encoding amino acids 597 to 613 with the comparable ³⁸ sequences (encoding amino acids 312 to 330) by PCR and standard cloning techniques .

The pACcI32 plasmid (27), which was used to construct cI-bait fusion proteins, contains a chloramphenicol resistance marker, a p15A replication origin, and a short alanine linker engineered into the cI protein gene such that the gene of interest can be fused to the 3' end of the cI protein by using NotI, AscI, BstYI, or BglII restriction sites . In this construction, the cI fusion is under the control of P_lacUV5 . Fragments to be cloned into pACcI32 were obtained as PCR products of pMot58 (to construct pcI-MotA^fl, pcI-MotA^NTD, and pcI-MotA^-) (23), pMot21 (to construct pcI-Mot21^NTD) (18), or pAsiA (to construct pcI-AsiA) (25) by using PfuTurbo DNA polymerase (Stratagene) and appropriate primers . Primers contained the necessary NotI or BglII sites to allow ligation with pACcI32 that had been previously cleaved with NotI and BglII . PCR products were purified by using a Wizard PCR purification system (Promega) and cloned into pACcI32 by using standard techniques . The ^1-570 gene was obtained as a PCR product of pJH62 (gift of V . J . Hernandez, State University of New York at Buffalo), a plasmid that contains the entire rpoD gene (encoding ⁷⁰) . PCR was performed with Amplitaq polymerase (Perkin-Elmer), a primer that annealed to the start of the ⁷⁰ gene and began with an XbaI recognition sequence, and a primer that annealed to sequences surrounding codon no . 571, introduced a stop codon of TAA at that position, and ended with a SacI recognition sequence . After cleavage with XbaI and SacI, the PCR product was ligated into pet21a(+) (Novagen) that had been previously cleaved with XbaI and SacI . The ClaI/SacI fragment, which contained the C-terminal region of the ⁷⁰ gene, was then obtained from the resulting plasmid . This fragment was used to replace the corresponding fragment in pLHN12 (22, 42), a plasmid that contains the wild-type ⁷⁰ gene downstream of a T7 promoter . The resulting plasmid was designated p^1-570 .

p^fl, which contains an N-terminal His-tagged ⁷⁰ gene, has been described previously (63) . This plasmid produces full-length ⁷⁰ with the amino acid sequence MRGSHHHHHHGSSGLVPRGSGLGTRL at its N terminus (^fl) . PCR products encompassing the C-terminal region of ^R596H and ^R608C were obtained from pBR-^R596H and pBR-^R608C, respectively, by using a primer that annealed at the ClaI site within the ⁷⁰ sequence and another primer that annealed just downstream of the ⁷⁰ gene and introduced a HindIII site . PCR products encompassing the C-terminal region of ⁷⁰ with a stop codon at amino acid position 608 or 604 were obtained by using pBR-⁷⁰, the same upstream primer, and a primer that introduced a TAA at amino acid position 608 or 604, respectively, and introduced a HindIII site . In each case, the products were digested with ClaI and HindIII and then ligated into p^fl that had been previously digested with ClaI and HindIII, resulting in p^R596H, p^R608C, p^608-613, and p^604-613 .

DNA sequence analyses (47) of the inserted sequences in each mutant ⁷⁰ plasmid confirmed that only the intended changes were present . In some cases, this sequencing was done at the Center for Agricultural Biotechnology, University of Maryland .

Proteins. AsiA and MotA were purified as described previously (25) . Wild-type ⁷⁰ and ^1-570 were purified from cultures of pLHN12/pLysS/BL21(DE3) and p^1-570/pLysS/BL21(DE3), respectively, as described previously (22) . ^fl, ^R596H, ^R608C, ^608-613, and ^604-613 were purified as described previously (63), except that cells were broken by sonication . E . coli core polymerase was purchased from Epicentre Technologies .

The C-terminal MotA fragment (MotA^{cloned CTD}), which contained MotA amino acids 105 to 211, was obtained as follows . BL21(DE3) cells containing a plasmid that expresses the MotA^{cloned CTD} gene under the control of the T7 promoter 10 (15) (plasmid was the gift of M . Finnin, S . Porter, and S . White, St . Jude's Children's Hospital, Memphis, Tenn.) were grown in L broth plus 25 µg of kanamycin/ml at 37°C to mid-log phase . The synthesis of MotA^{cloned CTD} was induced by the addition of IPTG (isopropyl-ß-D-thiogalactopyranoside) to 1 mM . Cells were harvested after 2 h and broken by sonication, and highly purified MotA^{cloned CTD} was obtained after phosphocellulose chromatography as described previously (25), except that proteins were eluted from the column by steps of 0.2, 0.5, and 1 M NaCl in sonication buffer . The 0.5 M NaCl eluate, which contained the bulk of the C-terminal MotA protein, was used for subsequent experiments . A control fraction was obtained by the same procedure, except that the cells used were not induced .

Proteolyzed MotA protein (MotA^{proteolyzed CTD}) generated with endogenous proteases as a partially purified fraction of the wild-type protein (0.8 M phosphocellulose fraction) (23) was loaded onto a phosphocellulose column . Fractions containing the 13.5-kDa MotA^{proteolyzed CTD} eluted with a higher salt concentration (peak fractions eluting with 0.47 M NaCl) than that of the wild-type protein (peak fraction eluting with 0.4 M NaCl) . To obtain an N-terminal analysis of the proteolyzed fragment, the 13.5-kDa peptide was purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (31) and then transferred to a polyvinylidene difluoride membrane (Novex) by using a Novex Western transfer apparatus and a buffer of 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11), 1 mM EDTA, and 10% methanol . N-terminal sequence analysis (16 cycles performed at the W . M . Keck Foundation Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Yale University) indicated that the 13.5-kDa gel band was composed of approximately equal amounts of two proteins . One protein matched the amino acid sequence of MotA starting at amino acid 102 . The other matched the amino acid sequence of E . coli 50S ribosomal protein L24 .

The concentrations of MotA^{proteolyzed CTD} and MotA^{cloned CTD} were estimated after SDS-polyacrylamide gel electrophoresis by comparing these proteins with a known amount of wild-type MotA . Levels were corrected for the fact that the MotA fragments were one-half the size of the wild type and that in the case of MotA^{proteolyzed CTD}, only one-half of the band seen on the gel corresponded to the MotA peptide .

ß-Galactosidase assays. ß-Galactosidase assays were performed by a modification of the procedure of Jain (28) . Because the synthesis of either AsiA or MotA is toxic for E . coli (25), overnight cultures were always started from single colonies obtained by streaking the -80°C culture stock onto Luria-Bertani plates containing 50 µg of kanamycin/ml, 50 µg of carbenicillin/ml, and 25 µg of chloramphenicol/ml . The plates were then incubated at 37°C, and single colonies were used to inoculate liquid cultures of M9 plus Casamino Acids (Quality Biological) supplemented with the same antibiotics . Cultures were grown overnight at 37°C with aeration, diluted 1:100 in fresh media plus antibiotics, and then grown to mid-log phase . IPTG either was present throughout growth (for the assays in Fig . 3) or was added at the indicated concentration once the cells reached mid-log phase (for the assays in Fig . 4 and 5), and then the cells were grown for another 60 min . A final optical density at 600 nm (OD_600-final) of the cells was determined, and the cell pellets from 5 ml of culture were harvested by centrifugation at 1,880 x g at 4°C and then resuspended on ice in 0.2 ml of lacZ buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM 2-mercaptoethanol) . Cell extracts, obtained after the addition of 20 µl each of 0.1% (wt/vol) SDS and CHCl₃ and vigorous vortexing of the cell suspension for 10 s, were kept on ice until needed . A 20-µl aliquot of the extract was then added to 0.98 ml of the lacZ buffer, and the mixture was incubated for 5 min at 28°C . The reaction was started by the addition of 0.2 ml of a solution containing 4 mg of o-nitrophenyl-ß-D-galactopyranoside (ONPG) per ml of lacZ buffer . ß-Galactosidase activity was measured by the hydrolysis of ONPG . After the solution turned yellow, the reaction was stopped by the addition of 0.5 ml of 1 M Na₂CO₃, and the time needed for the reaction (T) was noted . Reaction mixtures were briefly vortexed and then centrifuged at 830 x g . The OD₄₂₀ of the clear supernatant was then measured . The ß-galactosidase activity in Miller units (40) was determined as (2,000 x OD₄₂₀)/(T x OD_600-final) .

 
Relative ß-galactosidase activities at a 100 µM IPTG concentration (Fig . 5) were calculated as follows:

Native protein gels. Protein complexes were assayed by electrophoresis on native polyacrylamide gels as described previously (18) .

In vitro transcriptions. Incubation buffer I contained 44 mM Tris-Cl (pH 8), 50 mM NaCl, 42% glycerol, 1 mM EDTA, 0.18 mM dithiothreitol, 0.008% Triton X-100, and 0.2 mM 2-mercaptoethanol . Incubation buffer II contained 5 mM Tris-Cl (pH 7.5), 69 mM Tris-acetate (pH 7.9), 25 mM NaCl, 5% glycerol, 0.18 mM EDTA, 0.27 mM dithiothreitol, 260 mM potassium glutamate, 6.9 mM magnesium acetate, and 173 µg of bovine serum albumin/ml . DNA buffer contained 7.4 mM Tris-Cl (pH 7.9), 51 mM Tris-acetate (pH 7.9), 57 mM NaCl, 2.1% glycerol, 0.66 mM EDTA, 0.13 mM dithiothreitol, 0.21 mM 2-mercaptoethanol, 190 mM potassium glutamate, 5.1 mM magnesium acetate, 130 µg of bovine serum albumin/ml, 220 µM ATP, 220 µM GTP, 220 µM CTP, and 11 µM [-³²P]UTP (6.5 x 10⁵ dpm/pmol) . Finally, 1x Tris-borate-EDTA (TBE) contained 2.5 mM EDTA and 89 mM Tris-borate (pH 8.3) .

Proteins were preincubated and mixed with the DNA template and other transcription components as indicated in the figure legend . Reaction mixtures were then placed at 37°C for 20 s before the addition of 0.5 µl of rifampin at 300 µg/ml . After an additional 7.5 min at 37°C, reaction mixtures were collected on dry ice . Twenty-five microliters of gel loading solution (1x TBE containing 7 M urea, 0.1% bromophenol blue, and 0.1% xylene cyanol FF) was added, and the samples were heated at 95°C for 2 min . An 8-µl sample was then subjected to electrophoresis in a 4% polyacrylamide, 7 M urea denaturing gel run in 0.5x TBE .

Protein-DNA gel retardation assays. Gel retardation assays were performed by using the procedure of Hinton (23) . K_D(app) was determined as the protein concentration needed to retard 50% of a 5'-³²P-labeled 74-bp fragment containing P_uvsX in a 10-µl reaction mixture that contained 0.05 pmol of P_uvsX DNA and 1.25 pmol of P_tac competitor DNA .

Quantitation of autoradiograms. After autoradiography, films were scanned with a Scanmaster III Plus from Howtek, Inc . Quantification was performed with Diversity One software from Protein-Databases, Inc .

 
The C-terminal region of ⁷⁰ interacts with the N-terminal domain of MotA in an E . coli two-hybrid assay. To investigate further the possibility of an interaction between MotA and the C-terminal region of ⁷⁰, we used an E . coli-based two-hybrid system (11, 27) . In this system (Fig . 2), a chromosomal reporter lacZ gene lies downstream of a promoter that has a binding site for cI protein at position -62 . The bait is created by the fusion of a protein or domain of interest to the 3' end of cI . The prey consists of ⁷⁰ amino acids 528 to 613, which start within region 3.2 and then include all of region 4 plus the far-C-terminal region (35) . This prey is fused in frame to the NTD of the subunit of RNA polymerase . The addition of IPTG induces the synthesis of both the -⁷⁰ chimera and the cI-bait protein . An interaction between the bait protein positioned at -62 and the C-terminal region of ⁷⁰, which is available in the pool of RNA polymerase that contains the -⁷⁰ chimera, then increases lacZ transcription .

 
As has been previously reported (10), there was a large increase in ß-galactosidase activity upon the addition of IPTG to cells containing pcI-AsiA and pBR-⁷⁰ (Fig . 3) . To assay an interaction between MotA and the ⁷⁰ prey, we tested a bait consisting of cI fused to the entire MotA gene (cI-MotA^fl) and a bait in which the motA gene was positioned out of frame with cI (cI-MotA^-) . The addition of IPTG resulted in an increase in the level of ß-galactosidase activity in cells containing the cI-MotA^fl fusion compared to the activity seen in the presence of cI-MotA^- or cI alone (Fig . 3) . Although this was only a twofold increase over background, it was highly reproducible . In addition, in control assays, cells containing pcI-MotA but lacking pBR-⁷⁰ expressed background levels of lacZ (data not shown), indicating that by itself pcI-MotA was not responsible for the increase in ß-galactosidase activity .

Residues within the NTD of MotA are required for MotA activation (14, 18) . Induced synthesis of a cI-MotA^NTD fusion, which contained MotA amino acids 1 to 97, in cells containing the -⁷⁰ chimera resulted in a significant increase in ß-galactosidase activity (Fig . 3) . As a control, we constructed a plasmid containing a fusion of cI with the comparable NTD of Mot21, a mutant of MotA in which the first 8 amino acids of MotA have been replaced with 11 different amino acids . Mot21 is a positive control mutant of MotA (18) . Full-length Mot21 binds DNA like wild-type MotA but fails to activate transcription or form a complex with ⁷⁰ in a native protein gel (18) . Production of cI-Mot21^NTD in cells containing the -⁷⁰ chimera resulted in a ß-galactosidase activity curve that was coincident with that observed with cI alone (Fig . 3) . In addition, the presence of pcI-MotA^NTD alone (in the absence of pBR-⁷⁰) resulted only in background levels of ß-galactosidase activity (data not shown) . Taken together, these results suggest that MotA interacts with the C-terminal region of ⁷⁰ and that the NTD of MotA is sufficient for this interaction .

The MotA^NTD-⁷⁰ interaction in the two-hybrid assay involves the far-C-terminal region of ⁷⁰. ³⁸, an alternative sigma factor for E . coli RNA polymerase that is used during stationary phase and under certain conditions of stress (58; reviewed in references 20 and 21), has a region 4 which is similar in amino acid sequence to that of ⁷⁰ (35) . Replacement of the ⁷⁰ residues in the -⁷⁰ chimera with the comparable region of ³⁸ (amino acids 243 to 330) resulted in background levels of ß-galactosidase activity when tested with either cI-MotA^NTD or cI-MotA^fl but resulted in nearly 600 Miller units of ß-galactosidase activity when tested with cI-AsiA (Fig . 4) . Furthermore, an -⁷⁰/³⁸ hybrid chimera, in which only the last 17 amino acids of ⁷⁰ (597 to 613) were replaced with comparable ³⁸ residues (amino acids 312 to 330), also gave background levels of ß-galactosidase activity with cI-MotA^NTD (Fig . 5) . In contrast, this chimera gave only twofold less activity with the cI-AsiA fusion than did the -⁷⁰ chimera (Fig . 5) . These results suggest that the far-C-terminal region of ⁷⁰ is involved in the ⁷⁰-MotA interaction .

To investigate the need for specific ⁷⁰ C-terminal residues, we tested -⁷⁰ chimeras containing the single amino acid substitutions R596H, H600A, H600R, or R608C in the ⁷⁰ moiety (Fig . 5) . The R596H, H600A, and H600R substitutions had little effect on the level of ß-galactosidase activity observed with either cI-MotA^NTD or cI-AsiA . In addition, the R608C substitution had no significant effect on the level of ß-galactosidase activity observed with cI-AsiA . However, this substitution caused a significant defect when the mutant chimera was tested with cI-MotA^NTD . Varying the IPTG concentration from 5 µM to 1 mM gave results similar to those obtained with 100 mM IPTG (data not shown), indicating that the low level of ß-galactosidase activity observed with cI-MotA^NTD and -^R608C could not be improved by increasing the levels of these proteins . In summary, these two-hybrid assays suggested that a residue(s) within the far-C-terminal region of ⁷⁰ is important for an interaction with MotA but is relatively unimportant for the ⁷⁰-AsiA interaction .

Polymerase reconstituted with a ⁷⁰ lacking either amino acids 608 to 613 or amino acids 604 to 613 is defective for MotA-dependent transcription in vitro. To investigate the involvement of the far-C-terminal amino acids of ⁷⁰ in MotA-dependent transcription, we tested ⁷⁰ mutant proteins in single-round in vitro transcription reactions by using a DNA template containing the T4 middle promoter P_uvsX . Control reactions contained either wild-type ⁷⁰ (^fl) or ^R596H, a ⁷⁰ whose mutation had no effect in the two-hybrid assays . With both of these ⁷⁰s, transcription from P_uvsX was observed in the presence of polymerase alone, was inhibited by the addition of AsiA, and was greatly activated by MotA/AsiA (Fig . 6 and data not shown) . MotA alone resulted in a small (approximately twofold) increase (Fig . 6 and data not shown) . Previous work has indicated that MotA alone has no effect on P_uvsX transcription in multiple-round transcription reactions (24) but gives this small effect in single-round reactions (D . M . Hinton, unpublished data) .

 
Despite the fact that in the two-hybrid assays the ⁷⁰ R608C substitution behaved as if it impaired the ⁷⁰-MotA interaction, polymerase containing this mutation was fully active in the in vitro transcription reactions (data not shown) . This result suggested either that the MotA-⁷⁰ interaction inferred from the two-hybrid assays was not relevant or that in the context of the transcription complex, this single mutation was not sufficient to impair MotA activity . Thus, we investigated whether ⁷⁰ C-terminal deletions of 6 (^608-613) or 10 (^604-613) amino acids affected transcription from P_uvsX . Although the level of transcription was lower than that of the wild type, polymerase containing either deletion was able to transcribe from P_uvsX in the absence of MotA/AsiA . Thus, the deletions did not destroy polymerase activity (Fig . 6A) . When polymerase with ^608-613 or ^604-613 was used, AsiA alone inhibited transcription significantly or partially, respectively (Fig . 6) . Thus, polymerases with these deletions were still susceptible to AsiA inhibition, although the larger deletion was more resistant to AsiA action . In contrast, polymerase with either ⁷⁰ deletion was significantly impaired in MotA/AsiA activation at P_uvsX (Fig . 6) . The transcription results support the idea that the far-C-terminal region of ⁷⁰ is important for MotA to function effectively as an activator . We speculate that the single R608C substitution was not sufficient to interfere with MotA activation in vitro because other stabilizing contacts within the transcription complex compensated for this mutation .

The CTD of MotA binds DNA. We tested MotA^{cloned CTD}, a protein containing amino acids 105 to 211, and MotA^{proteolyzed CTD}, a proteolyzed fraction of MotA starting at amino acid 102, for their abilities to bind a DNA fragment containing the T4 middle promoter P_uvsX in a gel retardation assay . Both peptides bound P_uvsX DNA (Fig . 7 and data not shown) . The K_D(app) determined for MotA^{proteolyzed CTD} was 400 nM, a value that is similar to the previously reported values of 220 nM (6) and 130 nM (52) for wild-type MotA . For MotA^{cloned CTD}, the determined K_D(app) was fourfold higher (2,000 nM) . These results suggest that the CTD of MotA is sufficient to bind DNA . As expected, MotA^{proteolyzed CTD}, which lacks the MotA^NTD that is required for activation, did not support activated transcription from P_uvsX in vitro (data not shown) .

 
Bacteriophage T4 middle promoters represent a hybrid of host and phage promoter sequences, having an excellent match to the ⁷⁰ -10 recognition sequence but lacking a good match to ⁷⁰ recognition sequences at -35 (3, 19, 38) . It is common for a promoter that lacks canonical -35 sequences to require an activator(s) for transcription initiation by RNA polymerase . Such activators fall into two general categories (reviewed in references 4, 26, and 46) . Class I activators bind to sites located upstream of the promoter sequences (at -61.5 or farther), while class II activators bind to sites centered near position -41.5, immediately adjacent to core promoter sequences . In both cases, however, these proteins appear to work by contacting polymerase directly and stabilizing the interaction of ⁷⁰ region 4.2 with noncanonical sequences within the -35 region of the promoter (2, 11, 30; reviewed in references 4, 26, and 46) .

Evidence indicates that MotA/AsiA-dependent activation does not fit either class I or class II . The MotA binding site lies within the core promoter sequence rather than adjacent to it or farther upstream (3, 19, 38) . In addition, within the MotA-AsiA-RNA polymerase-middle promoter complex, ⁷⁰ retains its contacts with the -10 region of the DNA, but the upstream protein-promoter contacts are significantly rearranged (1, 24) . We have previously proposed a model to explain this middle promoter architecture (52) . In this model (Fig . 8), AsiA interacts with residues within region 4 of ⁷⁰ (8, 49, 50, 53, 59), MotA binds to the MotA box (23, 48) and interacts with ⁷⁰ (18), and ⁷⁰ region 2.4 retains its contacts with the -10 element of the promoter DNA (24) . We speculated that positioning an interaction between MotA and the C-terminal region of ⁷⁰ would be reasonable, given that residues within region 4.2 of ⁷⁰ normally interact with the -35 sequences of the DNA and that the MotA binding site is centered at -30 (52) . The work here demonstrates that the NTD of MotA, which contains residues needed for activation (14, 18), can interact with the C-terminal region of ⁷⁰ . In addition, we have shown that a C-terminal peptide of MotA starting at amino acid 102 can bind DNA with a binding constant similar to that of the full-length protein . These results are consistent with the idea that the two physical domains of MotA (15), an NTD that is formed by five -helices and a short ß-ribbon (34) and a CTD that is composed of three -helices interspersed with six ß-strands (33), represent two functionally distinct domains . We suggest that MotA belongs to a class of activators that is distinct from both class I and class II . Instead of stabilizing the typical contacts between region 4.2 of ⁷⁰ and the DNA, an activator in this third class functionally replaces such contacts by serving as a molecular bridge between ⁷⁰ and the DNA .

 
The amino acid sequence of region 4 of ³⁸ is similar to that of region 4 of ⁷⁰ (35) and recognizes the same -35 canonical promoter element (13, 16) . We found that AsiA interacted with the C-terminal region of ³⁸ in the two-hybrid assay . AsiA binds to a broad surface of ⁷⁰ region 4.2 (8), and AsiA can tolerate amino acid changes throughout this binding surface (41) . In addition, in the two-hybrid assay, the AsiA-⁷⁰ interaction was not significantly affected by mutations at residues R596, H600, or R608 (reference 10 and this paper) . Thus, the simple explanation for an AsiA-³⁸ interaction is that this interaction occurs because region 4.2 of ³⁸ is very similar to that of ⁷⁰ . However, this result is surprising, since AsiA neither inhibits transcription by polymerase containing ³⁸ nor forms a complex with full-length ³⁸ in a native protein gel (8) . Our results suggest that AsiA is indeed able to interact with the C-terminal region of ³⁸ but that some feature of the full-length protein prevents this interaction .

In contrast to the results seen with AsiA, replacement of even the last 17 amino acids of ⁷⁰ with comparable ³⁸ residues eliminated the stimulation of ß-galactosidase activity observed with cI-MotA^NTD . This effect is specific for MotA, since the ⁷⁰/³⁸ chimera worked both with cI-AsiA (this paper) and with a cI that had been fused to the E . coli anti-sigma protein Rsd (S . L . Dove and A . Hochschild, unpublished data) . In addition, a substitution of R608C in -⁷⁰ also reduced the interaction with cI-MotA^NTD but had no effect on the interaction with cI-AsiA . However, not all substitutions within the C-terminal region of ⁷⁰ weakened the interaction of ⁷⁰ with cI-MotA^NTD . In particular, a substitution at R596 or H600 had no significant effects . In contrast, the substitution R596H significantly reduced the interaction between the fused ⁷⁰ moiety and either Rsd from E . coli or the related regulator, AlgQ, from Pseudomonas aeruginosa (10) . The specific effects of these various substitutions argue that the defects seen with cI-MotA^NTD and the mutant -⁷⁰ chimeras arise from a loss of the MotA-⁷⁰ interaction rather than a misfolding of the mutant chimeras . Furthermore, our in vitro transcription experiments indicated that a ⁷⁰ lacking 6 or 10 C-terminal amino acids is defective for MotA activation of transcription . Taken together, our results are consistent with the idea that the far-C-terminal region of ⁷⁰ contacts MotA and that this contact is necessary for MotA to work as an activator . Previous work has indicated that an amino acid substitution at position 604 can partially suppress the growth defect of a T4 motA-positive control mutant in vivo (7), a result that is compatible with this conclusion .

The far-C-terminal region of ⁷⁰ lies within an alpha helix (amino acids 603 to 613) (5, 36) at the very end of the protein . This region is just C-terminal of residues that interact with the -35 region of DNA (residues 584, 585, and 588) (5, 9, 17, 29, 54) and of residues that have been implicated in the interactions of ⁷⁰ with E . coli class II activators (residues 590 to 603) (32, 36) . Thus, the C terminus of ⁷⁰ appears to be involved both in class II activation and in the architecturally different activation achieved by MotA/AsiA . Further studies will be needed to determine exactly how the region is configured in class II- versus MotA/AsiA-dependent activation .

This work was supported in part by National Institutes of Health grant GM44025, an established investigatorship from the American Heart Association (to A.H.), and a Charles A . King Trust postdoctoral fellowship (to S.L.D.) .

Present address: SHU-CHI LLC, Fairfax, VA 22031 .

Present address: Division of Infectious Diseases, The Children's Hospital, Harvard Medical School, Boston, MA 02115 .

1. Adelman, K., E . Brody, and M . Buckle. 1998 . Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle . Proc . Natl . Acad . Sci . USA 95:15247-15252.
2. Bown, J., A . Kolb, C . Meares, A . Ishihama, S . Minchin, and S . Busby. 2000 . Positioning of region 4 of the Escherichia coli RNA polymerase ⁷⁰ subunit by a transcription activator . J . Bacteriol . 182:2982-2984.
3. Brody, E., D . Rabussay, and D . Hall. 1983 . Regulation of transcription of prereplicative genes, p . 174-183 . In C . K . Mathews, E . M . Kutter, G . Mosig, and P . B . Berget (ed.), Bacteriophage T4 . American Society for Microbiology, Washington, D.C.
4. Busby, S., and R . Ebright. 1999 . Transcription activation by catabolite activator protein (CAP) . J . Mol . Biol . 293:199-213.
5. Campbell, E . A., O . Muzzin, M . Chlenov, J . L . Sun, C . A . Olson, O . Weinman, M . L . Trester-Zedlitz, and S . A . Darst. 2002 . Structure of the bacterial RNA polymerase promoter specificity sigma subunit . Mol . Cell 9:527-539.
6. Cicero, M., K . Alexander, and K . Kreuzer. 1998 . The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer . Biochemistry 37:4977-4984.
7. Cicero, M., M . Sharp, C . Gross, and K . Kreuzer. 2001 . Substitutions in bacteriophage T4 AsiA and Escherichia coli ⁷⁰ that suppress T4 motA activation mutations . J . Bacteriol . 183:2289-2297.
8. Colland, F., G . Orsini, E . Brody, H . Buc, and A . Kolb. 1998 . The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters . Mol . Microbiol . 27:819-829.
9. Dombroski, A., W . Walter, M . Record, Jr., D . Siegele, and C . Gross. 1992 . Polypeptides containing highly conserved regions of transcription initiation factor ⁷⁰ exhibit specificity of binding to promoter DNA . Cell 70:501-512.
10. Dove, S., and A . Hochschild. 2001 . Bacterial two-hybrid analysis of interactions between region 4 of the ⁷⁰ subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa . J . Bacteriol . 183:6413-6421.
11. Dove, S., F . Huang, and A . Hochschild. 2000 . Mechanism for a transcriptional activator that works at the isomerization step . Proc . Natl . Acad . Sci . USA 97:13215-13220.
12. Dove, S., J . Joung, and A . Hochschild. 1997 . Activation of prokaryotic transcription through arbitrary protein-protein contacts . Nature 386:627-630.
13. Espinosa-Urgel, M., C . Chamizo, and A . Tormo. 1996 . A consensus structure for ^S-dependent promoters . Mol . Microbiol . 21:657-659.
14. Finnin, M., M . Cicero, C . Davies, S . Porter, S . White, and K . Kreuzer. 1997 . The activation domain of the MotA transcription factor from bacteriophage T4 . EMBO J . 16:1992-2003.
15. Finnin, M., D . Hoffman, K . Kreuzer, S . Porter, R . Schmidt, and S . White. 1993 . The MotA protein from bacteriophage T4 contains two domains . Preliminary structural analysis by X-ray diffraction and nuclear magnetic resonance . J . Mol . Biol . 232:301-304.
16. Gaal, T., W . Ross, S . T . Estrem, L . H . Nguyen, R . R . Burgess, and R . L . Gourse. 2001 . Promoter recognition and discrimination by E^S RNA polymerase . Mol . Microbiol . 42:939-954.
17. Gardella, T., H . Moyle, and M . Susskind. 1989 . A mutant Escherichia coli ⁷⁰ subunit of RNA polymerase with altered promoter specificity . J . Mol . Biol . 206:579-590.
18. Gerber, J., and D . Hinton. 1996 . An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription . J . Bacteriol . 178:6133-6139.
19. Guild, N., M . Gayle, T . Sweeney, T . Hollingsworth, T . Modeer, and L . Gold. 1988 . Transcriptional activation of bacteriophage T4 middle promoters by the motA protein . J . Mol . Biol . 199:241-258.
20. Hengge-Aronis, R. 1993 . Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E . coli . Cell 72:165-168.
21. Hengge-Aronis, R. 1996 . Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli . Mol . Microbiol . 21:887-893.
22. Hernandez, V., L . Hsu, and M . Cashel. 1996 . Conserved region 3 of Escherichia coli ⁷⁰ is implicated in the process of abortive transcription . J . Biol . Chem . 271:18775-18777.
23. Hinton, D. 1991 . Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase . J . Biol . Chem . 266:18034-18044.
24. Hinton, D., R . March-Amegadzie, J . Gerber, and M . Sharma. 1996 . Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a ⁷⁰ binding protein, in the formation of the open complex . J . Mol . Biol . 256:235-248.
25. Hinton, D., R . March-Amegadzie, J . Gerber, and M . Sharma. 1996 . The bacteriophage T4 middle transcription system: T4-modified RNA polymerase, AsiA (sigma-70 binding protein), and the transcriptional activator MotA . Methods Enzymol . 274:43-57.
26. Hochschild, A., and S . Dove. 1998 . Protein-protein contacts that activate and repress prokaryotic transcription . Cell 92:597-600.
27. Hu, J., M . Kornacker, and A . Hochschild. 2000 . Escherichia coli one- and two-hybrid systems for the analysis and identification of protein-protein interactions . Methods 20:80-94.
28. Jain, C. 1999 . An Escherichia coli-based genetic strategy for characterizing RNA binding proteins, p . 161-175 . In S . Haynes (ed.), RNA-protein interaction protocols . Humana Press, Totowa, N.J.
29. Keener, J., and M . Nomura. 1993 . Dominant lethal phenotype of a mutation in the -35 recognition region of Escherichia coli ⁷⁰ . Proc . Natl . Acad . Sci . USA 90:1751-1755.
30. Kuldell, N., and A . Hochschild. 1994 . Amino acid substitutions in the -35 recognition motif of ⁷⁰ that result in defects in phage repressor-stimulated transcription . J . Bacteriol . 176:2991-2998.
31. Laemmli, U. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227:680-685.
32. Li, M., H . Moyle, and M . Susskind. 1994 . Target of the transcriptional activation function of phage cI protein . Science 263:75-77.
33. Li, N., E . A . Sickmier, R . Zhang, A . Joachimiak, and S . W . White. 2002 . The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain: the ‘double wing’ motif . Mol . Microbiol . 43:1079-1088.
34. Li, N., W . Zhang, S . White, and R . Kriwacki. 2001 . Solution structure of the transcriptional activation domain of the bacteriophage T4 protein, MotA . Biochemistry 40:4293-4302.
35. Lonetto, M., M . Gribskov, and C . Gross. 1992 . The ⁷⁰ family: sequence conservation and evolutionary relationships . J . Bacteriol . 174:3843-3849.
36. Lonetto, M., V . Rhodius, K . Lamberg, P . Kiley, S . Busby, and C . Gross. 1998 . Identification of a contact site in region 4 of the Escherichia coli RNA polymerase ⁷⁰ subunit . J . Mol . Biol . 284:1353-1365.
37. March-Amegadzie, R., and D . M . Hinton. 1995 . The bacteriophage T4 middle promoter P_uvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription . Mol . Microbiol . 15:649-660.
38. Marshall, P., M . Sharma, and D . Hinton. 1999 . The bacteriophage T4 transcriptional activator MotA accepts various base pair changes within its binding sequence . J . Mol . Biol . 285:931-944.
39. Mattson, T., J . Richardson, and D . Goodin. 1974 . Mutant of bacteriophage T4D affecting expression of many early genes . Nature 250:48-50.
40. Miller, J. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
41. Minakin, L., J . Camarero, M . Holford, C . Parker, T . Muir, and K . Severinov. 2001 . Mapping the molecular interface between the ⁷⁰ subunit of E . coli RNA polymerase and T4 AsiA . J . Mol . Biol . 306:631-642.
42. Nguyen, L. 1996 . Ph.D . thesis . University of Wisconsin, Madison.
43. Ouhammouch, M., K . Adelman, S . Harvey, G . Orsini, and E . Brody. 1995 . Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters . Proc . Natl . Acad . Sci . USA 92:1415-1419.
44. Ouhammouch, M., G . Orsini, and E . Brody. 1994 . The asiA gene product of bacteriophage T4 is required for middle mode RNA synthesis . J . Bacteriol . 176:3956-3965.
45. Pahari, S., and D . Chatterji. 1997 . Interaction of bacteriophage T4 AsiA protein with Escherichia coli ⁷⁰ and its variant . FEBS Lett . 411:60-62.
46. Rhodius, V., and S . Busby. 1998 . Positive activation of gene expression . Curr . Opin . Microbiol . 1:152-159.
47. Sanger, F., S . Nicklen, and A . R . Coulson. 1977 . DNA sequencing with chain-terminating inhibitors . Proc . Natl . Acad . Sci . USA 74:5463-5467.
48. Schmidt, R . P., and K . N . Kreuzer. 1992 . Purified MotA protein binds the -30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro . J . Biol . Chem . 267:11399-11407.
49. Severinov, K., and T . Muir. 1998 . Expressed protein ligation, a novel method for studying protein-protein interactions in transcription . J . Biol . Chem . 273:16205-16209.
50. Severinova, E., K . Severinov, D . Fenyo, M . Marr, E . N . Brody, J . W . Roberts, B . T . Chait, and S . A . Darst. 1996 . Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit . J . Mol . Biol . 263:637-647.
51. Severinova, E., K . Severinov, and S . Darst. 1998 . Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA . J . Mol . Biol . 279:9-18.
52. Sharma, M., P . Marshall, and D . Hinton. 1999 . Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts . J . Mol . Biol