Ada Protein-RNA Polymerase sigma  Subunit Interaction and alpha  Subunit-Promoter DNA Interaction Are Necessary at Different Steps in Transcription Initiation at the Escherichia coli ada and aidB Promoters*

Paolo LandiniDagger §, Jonathan A. BownDagger , Michael R. Volkert, and Stephen J. W. BusbyDagger

From the Dagger  School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, and the  Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha  subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha  subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma 70 subunit (amino acids 574-613). Several negatively charged residues in the sigma 70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha  subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma 70 interaction.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Transcription activation is one of the principal strategies used by bacteria to respond to external stimuli and to adapt to a changing environment. Most Escherichia coli activators stimulate transcription by establishing protein-protein interaction with RNA polymerase. Different subunits of RNA polymerase can be a target for transcription activators; however, the majority of activators interact with either the alpha  or the sigma 70 subunits (1, 2). alpha  and sigma 70 are also the subunits of RNA polymerase responsible for specific binding to promoters; sigma 70 contacts the -35 and -10 promoter elements (core promoter elements), whereas alpha  interacts with UP elements. At the strong rrnB P1 promoter, an UP element stimulates transcription initiation 30-fold through direct interaction with the alpha  subunit C-terminal domain (alpha CTD),1 in the absence of any other protein factors (3).

Exposure of E. coli to sublethal concentrations of methylating agents such as methyl methanesulfonate (MMS) activates expression from three promoters: the ada promoter (which also controls expression of the alkB gene), the alkA promoter, and the aidB promoter. This process is called the adaptive response (4-7). The product of the ada gene, the Ada protein, plays a dual role in the adaptive response; Ada transfers methyl groups from DNA to two of its cysteine residues, thereby functioning as a DNA repair protein. Upon self-methylation, Ada is converted into an activator able to stimulate transcription of the adaptive response genes, including its own (8-10). Ada is a 39-kDa protein organized in two independently structured domains, each with one methyl-acceptor cysteine (11). Methylation of Cys-69, in the N-terminal domain, triggers specific DNA binding by Ada and is required for transcription activation. In contrast, Ada protein singly methylated at Cys-321 is not capable of specific DNA binding activity. However, the Ada C-terminal domain does play a role in transcription activation; deletions in Ada CTD affect transcription of ada, even though they do not affect specific DNA binding (12-14). The methylated Ada protein (meAda) recognizes the sequence AAT(N)6GCAA, which at ada and aidB is located 5 and 7 base pairs upstream of the -35 sequence, respectively (10, 15).

In the model for meAda activation previously proposed (16), meAda contacts RNA polymerase through protein-protein interaction with alpha CTD and recruits RNA polymerase to the promoter region (15, 16). However, in a previous report (17), it was shown that RNA polymerase binds the ada and aidB promoters via alpha CTD, regardless of the presence of Ada. In the absence of meAda, RNA polymerase binds to the -60 to -40 region, which also includes the meAda binding site. This region closely resembles the UP element of the rrnB P1 promoter in A/T content, in location, and in its function as a binding site for the alpha  subunit of RNA polymerase. Mutations in alpha CTD that abolish alpha  binding to the rrnB P1 UP element also affect binding to the -60 to -40 region of ada and aidB (17) as well as transcription activation by meAda (16, 18). Due to these similarities, the -60 to -40 regions of ada and aidB can be considered as "UP-like elements." UP-like elements promote RNA polymerase binding to the ada and aidB promoters in the absence of meAda, but the resulting RNA polymerase-promoter binary complexes can only initiate transcription with poor efficiency. Binding of meAda promotes the formation of a ternary complex that is proficient in transcription initiation (17).

In this report, we show that although alpha CTD is responsible for the formation of the initial RNA polymerase-promoter binary complex, transcription activation requires protein-protein interaction between meAda and the C-terminal domain of the sigma 70 factor of RNA polymerase (sigma 70 CTD). Our observations suggest that alpha  and sigma 70 CTD are necessary for different steps in transcription initiation at the ada and aidB promoters.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of aidB Hybrid Promoters and Promoter Activity in Vivo-- The minimal sequence requirement for Ada-dependent activation of the aidB promoter is shown in Fig. 1. This fragment was subcloned into the multiple cloning site of the plasmid pSL1180 (Amersham Pharmacia Biotech), producing plasmid pPL115. The substitutions of the -10 and -35 promoter elements to consensus sequences, and of the aidB -60 to -40 region to the rrnB P1 UP element (see "Results"), were introduced by using double strand oligodeoxynucleotides. Plasmid pPL115 was digested with either BamHI and NcoI or NcoI and MluI and religated in the presence of complementary double strand oligodeoxynucleotides carrying the desired substitutions and the corresponding restriction sites. The ligation mixtures were used to transform E. coli strain RB791 (19), and recombinant plasmids were sequenced using the T7 sequencing kit (Pharmacia). The promoters were then tested for their in vivo activity by beta -galactosidase assays in the rpoS strain MV2792. Deletion of the rpoS gene completely abolishes ada-independent regulation of aidB (20, 21). Strains were grown to 0.2 A600 nm in LB medium supplemented with 0.2% glucose, 20 µg/ml tetracycline, and 80 µg/ml ampicillin and then rediluted 1:50. At an A600 nm of about 0.02, the cultures were divided in two aliquots, and one was supplemented with 0.04% MMS to activate the adaptive response. Samples were taken 2 h after induction, and beta -galactosidase activity was measured as described in Ref. 22. For beta -galactosidase experiments with wild type and mutant rpoD alleles, strain MV3766 (alkB::lambda PSG1 camR lacZ) was used. beta -Galactosidase activity was measured as described above, except that 25 µg/ml chloramphenicol was added to the medium, and MMS induction was started at 0.1 A600 nm.

In Vitro Transcription-- Reconstitution of RNA polymerase with histidine-tagged full-length alpha  or histidine-tagged truncated alpha -235 was performed as in Ref. 23. No contamination from wild type alpha  was detectable by SDS-polyacrylamide gel electrophoresis in the alpha -235 RNA polymerase preparation. For reconstitution of RNA polymerase with wild type sigma  factor or the E575V mutant, histidine-tagged sigma  was purified using Ni-NTA columns (Quiagen), using the standard protocol provided by the manufacturer. Purified sigma  factors were added at a 4:1 ratio to core RNA polymerase (Epicentre). Ada was purified as in Ref. 14 and methylated prior to use by the method reported in Ref. 9; when necessary, 0.2 µM of meAda was added to the transcription reaction mixture. Single-round in vitro transcription experiments from linear templates were performed as in Ref. 18. 5 pmol (0.1 µM) of reconstituted RNA polymerases was used, except for alpha -235 RNA polymerase, when 12.5 pmol (0.25 µM) was used. For the experiments in Fig. 3, the DNA templates were EcoRI-ScaI fragments from pPL115 (wild type aidB promoter sequence) or from pPL116 (aidB derivative with a perfect consensus -10 sequence). Both fragments are 166 base pairs long and produce an RNA transcript of 40 nucleotides. A DNA fragment carrying the lacUV5 promoter was used as internal control. The fragment is 205 base pairs long and produces a transcript of 65 nucleotides (9). For the experiments in Fig. 8, the template was a HindIII-EcoRI fragment from pYN3066 for the ada promoter (9) and an EcoRI-ScaI fragment from pPL115, described above, for aidB. The amount of transcription was quantified after normalization to the lacUV5 transcript using a phosphoimager (Molecular Dynamics).

Gel Retardation Assays-- Fragments for gel retardation assays were the same as those used for the in vitro transcription experiments. Fragments were labeled, and 5,000 cpm/sample was used in 20 µl final volume of reaction buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2.5 mM dithiothreitol, 6.25% glycerol, 10 µg/ml herring sperm DNA). Wild type and mutant RNA polymerases, sigma  factors (purified as in Ref. 24), and meAda were added as described in the figure legends. Samples were incubated 20 min at 37 °C and loaded either on to a 4% (for experiments with RNA polymerase) or a 6% (for experiments with sigma  factors) native polyacrylamide gel. Gels were run at 10 V/cm in 0.25 × TBE (22.5 mM Tris borate, 0.5 mM EDTA), 1.25% glycerol. Bands were visualized by autoradiography.

Screening of rpoD Mutations-- To screen for rpoD mutant alleles affecting ada-dependent transcription, a plasmid library carrying mutations resulting in single alanine substitutions at 17 amino acids of sigma 70 C-terminal domain (obtained from C. Gross, University of California, San Francisco; plasmids are derivatives of pGEX-2Tsigma 70 (25)) was used to transform MV3766 (ada-alkB::lacZ). Strain MV3766 carries a lacZ transcriptional fusion in the alkB gene, which lies downstream of the ada gene and whose transcription is driven by the ada promoter. Additional mutagenesis of the terminal segment of the rpoD gene was performed by two independent polymerase chain reactions from an XhoI site (corresponding to amino acid 528 of sigma 70) to a HindIII site immediately downstream of the stop codon. Strain MV3766 was transformed with the plasmid library. Transformants were plated on LB medium and replica-plated onto MacConkey medium both in the presence and in the absence of 0.02% MMS. In the presence of MMS, colonies with normal levels of ada-dependent transcription are pink, whereas colonies with decreased levels are white. The replica-plating step is necessary to avoid exposure of the colonies to the mutagenic effects of MMS. Colonies corresponding to the mutant candidates were picked from the MacConkey plates with no MMS and tested for beta -galactosidase activity as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

In Vivo Transcription from aidB Promoter Derivatives-- Fig. 1 shows the sequence of the ada and aidB promoters. The -60 to -40 regions of the two promoters show a high degree of similarity to the UP element of the rrnB P1 promoter. This region is also the binding site for meAda, and its deletion abolishes ada-dependent activation (Ref. 26; Fig. 2). Although both promoters have recognizable -35 and -10 sequences, they differ from the consensus hexamers TTGACA and TATAAT. At positively controlled promoters, one of the functions of activator proteins is to improve recognition of one or more weak promoter elements by RNA polymerase through either protein-protein interaction or modification of local DNA structure. To understand which of the weak promoter elements is the target of meAda activation, we constructed a set of derivatives of the aidB promoter in which either the -35 or the -10 elements were substituted by perfect consensus hexamers, and the UP-like element was substituted by the UP element of rrnB P1. We investigated the effects of these substitutions on both basal and ada-activated levels of transcription in vivo.


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Fig. 1.   Sequence of the ada and aidB promoters. Numbering is relative to the transcription start. The -35 and -10 elements are shown in boldface type. The alpha -binding sites (UP-like elements) are underlined. The location of the restriction sites used for the introduction of the mutagenic oligonucleotides in aidB are italicized.


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Fig. 2.   In vivo transcription of aidB promoter derivatives (performed as in "Experimental Procedures"). Open bars, beta -galactosidase levels in the absence of MMS. Filled bars, beta -galactosidase levels in cells treated with 0.04% MMS. Data are the average of four independent experiments. The standard deviation was <15%.

Results shown in Fig. 2 suggest that at the rrnB UP element/aidB hybrid promoter (aidB "rrnB UP"), ada-independent transcription levels are only slightly higher than for the wild type aidB promoter (1.6-fold, Fig. 2). Thus, meAda does not activate transcription by converting the alpha  binding site of aidB into a better UP element. Because this substitution also replaces the Ada binding site, ada-dependent activation is almost completely abolished. Changes to consensus -35 or -10 sequences did have more pronounced effects on transcription; the consensus -10 increased ada-independent transcription levels by almost 5-fold, and introduction of a consensus -35 resulted in a more than 12-fold increase. However, neither substitution completely relieved the dependence on meAda for optimal promoter expression; meAda activates the aidB promoter derivative with a consensus -10 element (aidB "-10 con") by 5-fold, and the derivative with a consensus -35 element (aidB "-35 con") by 2.3-fold (Fig. 2). These observations suggest that meAda activates transcription by affecting RNA polymerase interaction with both the -35 and -10 elements of aidB.

In Vitro Transcription-- Sakumi et al. (16) had proposed that meAda activates transcription by direct contact with the CTD of the alpha  subunit. We further investigated the role of alpha CTD in activation by meAda by performing in vitro transcription experiments using two forms of reconstituted RNA polymerase different with respect to their alpha  subunits: one form carried wild type alpha , the other a mutant alpha  deleted of the C-terminal 94 amino acids (alpha -235). Although alpha -235 RNA polymerase is impaired in UP element utilization and in transcription from some activator-dependent promoters, it is proficient in transcription from factor-independent and promoters dependent on activators that do not interact with alpha  (1).

We tested the two forms of reconstituted RNA polymerase for transcription from the wild type aidB promoter (aidB wt) as well as from an otherwise identical promoter in which the -10 sequence was changed to consensus (aidB "-10 con"). The latter promoter shows 5-fold higher basal transcription level in vivo compared with the wild type aidB promoter but is still dependent on meAda for maximal promoter expression (Fig. 2). Deletion of the alpha CTD dramatically affects both meAda-dependent and independent transcription from the wild type aidB promoter, even though activation by meAda is not totally abolished (Fig. 3, lanes 5 and 6). Transcription from aidB "-10 con" by alpha -235 RNA polymerase is also affected; however, at this promoter meAda activates transcription by both forms of RNA polymerase to a similar extent (5.2-fold for wild type alpha - and 4-fold for alpha -235 RNA polymerase; Fig. 3, lanes 7-10). These results strongly suggest that, although alpha CTD is necessary for efficient transcription at the wild type aidB promoter, it is not required for activation by meAda.


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Fig. 3.   In vitro transcription with reconstituted wild type (0.1 µM) and alpha -235 (0.25 µM) RNA polymerases. The filled arrow head shows the position of the transcript from the lacUV5 promoter (placUV5). The open arrow head indicates the position of transcripts from either the wild type aidB promoter (paidB wt, lanes 3-6) or the aidB "-10 con" promoter (paidB -10 con, lanes 7-10). Fold-activation by meAda is shown below the transcripts.

The results of in vitro transcription experiments raise the possibility that alpha -235 RNA polymerase is not proficient in carrying out transcription from the wild type aidB promoter because it is affected in RNA polymerase-promoter interaction, rather than interaction with meAda. To investigate this possibility, we tested both wild type and alpha -235 RNA polymerase for their ability to bind the wild type aidB promoter in the absence of meAda. As shown in Fig. 4, wild type RNA polymerase binds aidB wt at 0.04-0.08 µM, whereas alpha -235 RNA polymerase fails to bind the promoter at concentrations up to 0.32 µM. A similar result was obtained for the wild type ada promoter (data not shown). Both forms of RNA polymerase were equally as efficient in binding both the lacUV5 and the galP1 promoters under the same experimental conditions (data not shown). The above results show that alpha CTD promotes recruitment of RNA polymerase to the ada and aidB promoters independently of meAda.


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Fig. 4.   Gel retardation experiments performed with wild type and alpha -235 forms of RNA polymerase. Lanes 1 and 6, wild type aidB promoter, no proteins added. Lanes 2-5, increasing concentrations of wild type RNA polymerase (0.04, 0.08, 0.16, 0.32 µM). Lanes 7-10, same concentrations of alpha -235 RNA polymerase. F indicates the free DNA fragment.

Gel Retardation Studies with meAda and sigma 70-- The location of meAda binding sites at the ada and aidB promoters is consistent with the possibility of interaction with the sigma 70 factor of RNA polymerase. To investigate this possibility, gel retardation experiments were performed with meAda and purified sigma 70. sigma 70 is capable of specific DNA binding only when assembled into RNA polymerase holoenzyme (27). Indeed, no binding of sigma 70 alone to either the ada or aidB promoters could be detected; however, addition of sigma 70 (0.5 µM) to a meAda·promoter complex resulted in a supershift at both the ada (Fig. 5) and the aidB (data not shown) promoters. This suggests that sigma 70 is able to form a ternary complex with meAda at either the ada or the aidB promoter DNA. The presence of multiple shifts is possibly due to partial dissociation of the meAda·DNA complex in the experimental conditions used and is a typical pattern for meAda (10). In control gel retardation experiments, no significant binding by meAda and sigma 70 was detected with unrelated DNA fragments; sigma 70 was unable to promote binding of the unmethylated Ada protein to the ada promoter. Finally, sigma 70 did not produce any supershift when added to a CRP·gal P1 promoter complex (data not shown).


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Fig. 5.   Gel retardation experiments with purified sigma 70 factor at the ada promoter (Pada). Lane 1, no proteins added; lane 2, sigma 70 (0.5 µM); lane 3, meAda (0.4 µM); lane 4, sigma 70+meAda. F indicates the free DNA fragment; CI indicates the meAda·DNA complexes; CII indicates the sigma 70/meAda·DNA complexes.

Gel retardation experiments were performed with two sigma 70 deletion mutants, sigma 574 and sigma 529; these mutants, deleted of 39 and 84 amino acids in the C-terminal domain, respectively, are impaired in interaction with some activators when assembled into RNA polymerase holoenzyme (24). Concentrations up to 5 µM of the two mutant sigma  factors promote little formation of ternary complexes at either the ada (Fig. 6) or the aidB (data not shown) promoter; in the same experiment, complete supershift of the two complexes occurred at 1 µM with wild type sigma 70.


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Fig. 6.   Gel retardation in the presence of meAda and wild type and truncated sigma  factors at the ada promoter (Pada). When present, concentration of meAda was 0.4 µM. F indicates the free DNA fragment; CI indicates the meAda·DNA complexes; CII indicates the sigma 70/meAda·DNA complexes.

Substitutions in sigma 70 Affect meAda-dependent Transcription-- The above gel retardation experiments suggest that the C-terminal domain of sigma 70 could be the target for activation by meAda. Therefore, we expected mutations in the terminal segment of the rpoD gene to specifically affect ada-dependent transcription. We screened for this class of rpoD mutants using a plasmid library carrying rpoD alleles in which the segment encoding for amino acids 528-613 of sigma 70 had been mutagenized by polymerase chain reaction. The plasmid library was used to transform strain MV3766. This strain carries a lacZ transcriptional fusion in the chromosomal alkB gene, whose transcription is driven by the ada promoter. Over 1700 colonies were screened and four mutant colonies were isolated. The mutant rpoD alleles were sequenced; three carried nonsense mutations, which resulted in truncations of sigma 70 at amino acid 560 (1 clone) or at amino acid 584 (2 clones). The fourth candidate carried a missense mutation, resulting in a glutamic acid to valine change at position 575 of sigma 70 (E575V).

In addition to polymerase chain reaction-directed mutagenesis, we also tested a set of plasmids (kindly given by C. Gross, UCSF) carrying rpoD alleles with single alanine substitutions at 17 amino acids of sigma 70CTD (Fig. 7). beta -Galactosidase assays were performed to quantify the effect of the alanine substitutions, as well as the E575V mutation, on transcription from the ada promoter. In strain MV3766, wild type sigma 70 from the chromosomal rpoD gene is normally expressed, and mutated sigma  factors are present in only a slight excess to wild type sigma 70 (data not shown). Nevertheless, expression of several mutant rpoD alleles resulted in altered levels of in vivo transcription at the ada promoter: E574A, E575V, I590A, E591A, E605A, and D612A substitutions significantly decreased ada-dependent transcription, with E575V having the most severe effect (Fig. 7). Some mutations, such as R596A, K597A, R608A, and D613A, resulted instead in an increased level of ada-dependent transcription (120-150%, Fig. 7).


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Fig. 7.   Effects of substitutions in sigma 70CTD on ada-dependent transcription in vivo. Values are an average of three independent experiments and are given as a percentage of the wild type. The average value for the wild type was 903 Miller units.

To verify that inhibition of ada-dependent transcription in vivo is indeed due to disruption of meAda-sigma 70 interaction, we purified both wild type and E575V sigma  factors and reconstituted RNA polymerase in vitro. As shown in Fig. 8, E575V sigma 70-RNA polymerase was able to carry out transcription from the lacUV5 promoter with the same efficiency as wild type sigma 70-RNA polymerase, which suggests that the E575V mutation does not affect either core enzyme-sigma 70 interaction or factor-independent transcription; however, meAda-dependent transcription by the mutant RNA polymerase at both ada (Fig. 8) and aidB (data not shown) was drastically impaired.


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Fig. 8.   In vitro transcription with RNA polymerases reconstituted with either wild type or E575V sigma  at the ada promoter. Fold-activation by meAda is shown below the transcripts.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In a previous report, we showed that RNA polymerase binds to the ada and aidB promoters via the alpha  subunit, independently of the Ada protein. Thus, meAda does not recruit RNA polymerase to the promoters but rather converts the RNA polymerase-promoter complex into a ternary complex proficient in transcription initiation (17). The location of the meAda binding site suggests the possibility of interactions with either alpha CTD or sigma ; alternatively, the mechanism for activation by meAda could involve turning the alpha -binding sites of ada and aidB into more efficient UP elements. Our experiments with hybrid promoters show that when the UP-like element of aidB is substituted by the rrnB P1 UP element, no significant stimulation of transcription occurs (Fig. 2). In contrast, changing either the -35 or -10 sequences of aidB to consensus results in a significant stimulation of ada-independent transcription in vivo, suggesting that meAda improves RNA polymerase interaction with the core promoter region (Fig. 2). Thus, meAda activates transcription either by improving initial binding of RNA polymerase to the -35 or by facilitating a later step in transcription initiation, such as isomerization to open complex.

Deletion of alpha CTD severely affects meAda-activated transcription (Ref. 16; Fig. 3) and prevents RNA polymerase binding to the ada and aidB promoters (Fig. 4). However, at an aidB derivative in which the -10 sequence was changed to consensus (aidB "-10 con"), meAda is able to activate transcription by alpha -235 RNA polymerase (Fig. 3) to roughly the same extent as wild type RNA polymerase. Thus, dependence on alpha CTD for transcription at Ada-dependent promoters can be by-passed by strengthening the core promoter, presumably by providing an alternative binding site for alpha -235 RNA polymerase that compensates for its loss of interaction with the UP-like element. Although it is possible that altering the -10 sequence of the aidB promoter also modifies the interaction between meAda and RNA polymerase, the results at the aidB -10 con promoter clearly demonstrate that meAda can activate transcription by RNA polymerase containing an alpha  subunit deleted of its CTD. Although we cannot rule out the possibility of direct alpha -meAda interaction, we propose that inefficient transcription by RNA polymerase deleted of its alpha CTD results from the loss of the alpha -UP-like element interaction and consequent inability of RNA polymerase to bind the promoter, rather than from lack of interaction with meAda.

Several lines of evidence show that meAda interacts with sigma 70. Gel retardation experiments (Figs. 5 and 6) indicate that the terminal 39 amino acids of sigma 70 are necessary for this interaction. This region contains determinants for the recognition of the -35 element (region 4.2), followed by the so-called "basic cluster" (28). Several amino acids in both region 4.2 and in the basic cluster have been found to be important for interaction with transcription activators, such as PhoB, bacteriophage lambda  cI protein, and FNR (29, 30).2 A set of substitutions between amino acids 570 and 580 of sigma 70 severely impairs transcription activation by PhoB at the pstS promoter (31) and also affects activation by cI at lambda  PRM promoter (30). Mutational analysis of sigma 70CTD suggests that meAda interacts with a set of negatively charged residues both in and downstream of region 4.2 (Fig. 7). With the exception of I590A, all the substitutions that significantly affect ada-dependent transcription involve negatively charged amino acids (Glu-574, Glu-575, Glu-591, Glu-605 and Asp-612); substitution to alanine of several positively charged residues (Arg-596, Lys-597, Arg-608) results in increased levels of ada-dependent transcription (Fig. 7), supporting the hypothesis that meAda interacts with a negatively charged patch in sigma 70 CTD. One of the residues important for meAda-sigma 70 interaction, glutamic acid at position 575 (Glu-575), also plays a role in transcription activation by PhoB (29), suggesting that the location of this residue allows interaction with different transcription activators. The Glu-575 residue does not seem to be necessary for activator-independent transcription; neither the E575V mutation described in this report nor the E575K mutation described in Ref. 29 has any effect on transcription from the lacUV5 promoter in vitro (Fig. 8), nor do they affect growth rates in any of the reporter strains tested (data not shown; Ref. 29).

The specificity of meAda-sigma 70 interaction, the location of its determinants in the terminal 39 amino acids of sigma 70, and the strong effect of the E575V mutation in vitro point to sigma 70CTD as the principal target of activation by meAda at the ada and aidB promoters. Interestingly, meAda is also able to activate transcription by sigma s-RNA polymerase at both promoters (21, 32). sigma s is an alternative sigma  factor, highly expressed in the stationary phase of bacterial growth (33). sigma s is homologous to sigma 70 in region 4.2 (28), and the amino acids important for meAda-dependent transcription are either conserved residues (Glu-574/Glu-289 and Ile-590/Ile-305) or conservative substitutions (Glu-575/Asp-290, Glu-591/Gln-306, and Glu-605/Gln-320) in sigma s. Thus, it is possible that meAda could contact amino acids conserved in both sigma  factors; future experiments of site-directed mutagenesis in sigma s will allow better understanding of activation of sigma s-dependent transcription by meAda.

A model for meAda-activation of ada and aidB is presented in Fig. 9. In the absence of meAda, RNA polymerase can bind to the ada and aidB promoters via its alpha CTD but fails to establish any strong interaction with the core promoter (17). meAda does not recruit RNA polymerase to the ada and aidB promoters, but upon binding of alpha  to the UP element it interacts with sigma 70 CTD, triggering transcription initiation; therefore, alpha  subunit-promoter and meAda-sigma 70 interactions act at separate but interdependent steps of transcription initiation. It is possible that Ada CTD is responsible for this interaction; deletions in Ada CTD abolish transcription activation at the ada promoter without affecting DNA binding by meAda (14).


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Fig. 9.   Model for transcription activation by meAda. The upper panel illustrates the specific interactions established between RNA polymerase and the ada or the aidB promoters in the absence of meAda; the RNA polymerase-promoter complex results from protein-DNA interactions between alpha CTD and the UP elements. The lower panel shows the RNA polymerase·promoter·meAda ternary complex. meAda binds to its DNA site via its N-terminal domain (NTD) (12) and stimulates transcription initiation (black arrow) via protein-protein interaction between its C-terminal domain (14) and sigma 70CTD.

    ACKNOWLEDGEMENTS

We thank Carol Gross for the gift of the alanine scan plasmid set, Alicia Dombroski for useful discussion, and Virgil Rhodius for providing us with the rpoD mutant library.

    FOOTNOTES

* This work was supported by a Long Term EMBO Fellowship and a TMR fellowship from the European Community (to P. L.).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. Tel.: 0044-121-414-5434; Fax: 0044-121-414-7366; E-mail: p.landini{at}bham.ac.uk.

1 The abbreviations used are: alpha CTD, alpha  subunit C-terminal domain; MMS, methyl methanesulfonate; meAda, the methylated form of the Ada protein.

2 M. Lonetto, V. Rhodius, K. Lamberg, P. Kiley, S. Busby, and C. Gross, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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