©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of puc Operon Expression in Rhodobacter sphaeroides
ANALYSIS OF THE CIS-ACTING DOWNSTREAM REGULATORY SEQUENCE (*)

(Received for publication, February 24, 1995; and in revised form, July 5, 1995)

Jeong K. Lee (§) Samuel Kaplan (¶)

From the Department of Microbiology and Molecular Genetics, University of Texas Houston Health Science Center, Houston, Texas 77225

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Both site-directed and spontaneous mutagenesis have been used to investigate the role of the cis-acting regulatory region between -92 and -1 base pair (bp) of the puc operon of Rhodobacter sphaeroides. The DNA sequence from -84 to -66 bp upstream of the 5` end of the start site of puc operon transcription is essential for normal puc operon expression. This regulatory effect was exerted irrespective of the presence or absence of additional upstream regulatory sequences extending from -629 to -93 bp. It is likely that this region is involved in activator binding. Additionally, two regions of dyad symmetry centered at -42 and -17 bp are shown to be involved in oxygen repression of puc operon expression. Mutations within these regions of dyad symmetry were further subdivided on the basis of whether or not the upstream regulatory region was required to observe the mutant phenotype. Based upon these observations we conclude that these regions of dyad symmetry possessing the motif TGT-N-ACA (where N represents any nucleotide) are involved in repressor binding with the puc operon promoter overlapping each of these dyad symmetries.


INTRODUCTION

The puc operon of Rhodobacter sphaeroides consists of two structural genes, pucB and pucA, encoding the beta- and alpha-polypeptides, respectively, of the B800-850 light harvesting complex and additional downstream open reading frame(s) that encode gene product(s) involved in the post-transcriptional control of expression and assembly of the beta- and alpha-polypeptides to form the B800-850 complex(1, 2) . In a related bacterium, Rhodobacter capsulatus, the puc operon has been reported to have a similar although not identical organization (3, 4) . Formation of the B800-850 complex is variable with respect to the fixed photosynthetic unit. Together with the fixed photosynthetic unit the B800-850 complex has been referred to as the variable photosynthetic unit, which can comprise over 50% of the protein found in the intracytoplasmic membrane(5, 6, 7, 8, 9) . Intracytoplasmic membrane formation is represented as a series of invaginations arising from and continuous with the cytoplasmic membrane following removal or lowering of the partial pressure of O(2) below threshold levels, approximately 2.5% relative to air(10) .

The puc operon gives rise to two (0.5- and 2.3-kilobase) transcripts, having a single transcription start site, both of which are severely affected by the presence or absence of O(2), and in the absence of O(2) the relative levels of each transcript are further modulated by light intensity(1, 11) . We have subdivided the 629-bp (^1)puc DNA upstream of the 5`-end (+1) of the puc transcripts into two functionally separable cis-acting domains, the upstream regulatory sequence (URS) (-629 to -93) involved in enhanced control of operon transcription by oxygen and light, and the downstream regulatory sequence (DRS) (-92 to -1) primarily involved in basal level aerobic transcription as well as anaerobic regulation of transcription(12, 13) . Interaction or communication between these two cis-acting regions was demonstrated based upon the transcriptional response to oxygen and light and the silencing of point mutations within the DRS when these two domains are uncoupled(12) . A region extending from -139 to -93 bp containing overlapping FNR- and IHF-binding sequences separates these two cis-acting regions and is suggested to play a role in mediating this interaction(12, 13, 14) . The IHF-binding sequence is involved in repression of puc operon transcription by oxygen as well as modulation of the transcription levels by incident light intensity(14) . Recently the gene encoding an FNR homologue in R. sphaeroides has been identified in our laboratory. (^2)

Recently Eraso and Kaplan (15) reported a new gene locus in R. sphaeroides, prrA, which was found to be involved in positive regulation of photosynthesis gene expression in response to anaerobiosis. The NH(2)-terminal portion of PrrA showed clear similarity to the highly conserved amino termini of response regulators of two-component regulatory systems mediating signal transduction in prokaryotes. PrrA was shown to act (directly or indirectly) at the level of the DRS of the puc regulatory region. In R. capsulatus a gene designated regA was reported(16) , to which prrA shows a high similarity in both DNA and amino acid sequences. In the present studies, both in vitro and in vivo mutagenesis were employed to investigate the role of the DRS in puc operon expression. As a result, the DRS was itself subdivided into several regulatory regions. One of these domains is similar to those reported to be involved in factor-dependent binding(17) . A model is presented that describes the role(s) for each of these regions within the DRS in the regulation of puc operon expression.


EXPERIMENTAL PROCEDURES

Bacteria, Plasmids, and Cell Growth

The bacterial strains and plasmids used in this study are presented in Table 1. R. sphaeroides wild type strain 2.4.1 and its derivatives were grown as described previously(18) . R. sphaeroides cultures used for the measurement of beta-galactosidase activities were grown chemoheterotrophically or photoheterotrophically by sparging with gas phase mixtures as described previously(19) . Growth of R. sphaeroides cultures in the dark with Me(2)SO was performed as described previously(14) . For isolation of single colonies under photoheterotrophic (100 W/m^2) conditions, agar-embedded cultures in screw-cap tubes were employed.



Escherichia coli strains JM109, DH5alphaphe, TG1, or S17-1 were grown at 37 °C in Luria medium(20) . Ampicillin, tetracycline, kanamycin, streptomycin, and spectinomycin (final concentrations, 50, 20, 25, 50, and 50 µg/ml, respectively) were added to the growth medium for E. coli strains carrying plasmids encoding these drug resistance genes. Plasmids pUC19, pBS, pSL301, pRS415(21) , and pLV106 (12) were used for cloning.

DNA Manipulation and Sequence Analysis

Large and small scale plasmid DNA preparations were prepared as described previously (11, 20, 22) . Treatment of DNA with restriction enzymes and other nucleic acid-modifying enzymes was performed in accordance with manufacturer specifications. DNA fragments were analyzed on agarose gels or polyacrylamide gels, and restriction fragments were isolated as described previously(23) .

DNA sequence analysis was performed with phage M13 clones as described previously (11, 18) or with an ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Foster City, CA) at the DNA Core Facility of the Department of Microbiology and Molecular Genetics (University of Texas Health Science Center, Houston, TX).

Conjugation Techniques

Plasmid pLV106- or pCF1010-derived plasmids were mobilized into R. sphaeroides 2.4.1 by previously described procedures(19) .

Site-directed Mutagenesis of the puc Regulatory DNA

The oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.) was used according to the manufacturer's specifications. The presence of the desired mutations was confirmed by dideoxy sequencing of the mutated DNA molecules.

Construction of pCF1010 and Cloning of the puc::lacZ Transcriptional Fusion Constructs

pCF1010 was constructed from pLV106 through a series of clonings. The vector contains seven unique restriction sites (PstI, NotI, NsiI, AvrII, StuI, BspMII, and XbaI sites derived from pSL301) situated between a 2.0-kilobase cartridge harboring a spectinomycin/streptomycin-resistance gene (from pHP45) and a 5.1-kilobase DNA fragment containing lacZYA` (from pRS415). Transcriptional fusions between puc and lacZ within the vector can be performed by one-step cloning of a puc regulatory DNA fragment (from pUC19, pBS, or M13 clones) into the multiple cloning site of pCF1010. Cloning of the puc upstream DNA fragments to form transcriptional fusions of puc to lacZ within the restriction sites of pLV106 was performed as described(12) .

Preparation of Cell Extracts and Assay of beta-Galactosidase

Cell harvest, preparation of crude extracts, and beta-galactosidase assays were performed as described(24) . All experiments involving beta-galactosidase assays were conducted at least two times.

Materials

Restriction endonucleases and nucleic acid-modifying enzymes were purchased from Life Technologies, Inc., or New England Biolabs, Inc. (Beverly, MA) and used as specified by the manufacturer. The Klenow fragment of E. coli DNA polymerase I, proteinase K, and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside were obtained from Boehringer Mannheim. [alpha-S]dATP was obtained from Amersham Corp. Isopropyl-beta-D-thiogalactopyranoside and o-nitrophenyl beta-D-galactopyranoside were obtained from Sigma. Oligonucleotides used for construction of mutants were made by Genosys Biotechnologies, Inc. (The Woodlands, TX) or by the Microbiology and Molecular Genetics Core Facility (University of Texas Health Science Center, Houston, TX). DNA sequencing kits and Sequenase were obtained from U.S. Biochemical Corp. Molecular biology grade phenol was purchased from Fisher. All other chemicals used in this work were reagent grade.


RESULTS

A Putative Binding Site for a Transcription Activator in the DRS

Some general features of the cis-acting regulatory region involved in puc operon expression are presented in Fig. 1A. The importance of the 35-bp sequence, immediately downstream of the IHF-binding sequence(14) , between the XmaIII(-92) and HaeIII(-57) restriction sites (Fig. 1B) has been recognized previously (12) . A similar sequence and spacing to that found in R. sphaeroides (TGGC-N-TCGCA) is observed in R. capsulatus, TGGC-N(8)-CCGCA, and is located between -80 and -40 bp upstream of the 5`-ends of the puc transcripts(25, 26) . To further assess the role of this sequence in puc operon expression several alterations in this sequence were introduced by site-directed mutagenesis, and the results of these changes were examined by following the expression of lacZ transcription fusions.


Figure 1: A, restriction map of the puc upstream DNA. +1 is the 5`-end of the puc-specific transcripts. The DNA region corresponding to the sequence shown in B is underlined. B, DNA sequence from -148 to +1. An IHF-binding sequence (b) overlapping a putative consensus sequence for FNR (a) is followed by the putative activator-binding site(s) (c). The two regions of dyad symmetry are distal (d) and proximal (e).



In the first constructions, two consecutive bases, GG(-83, -82) or GC(-68, -67) were changed to TT and TA, respectively. Each of the altered DNA fragments corresponding to the DRS (-92 to -1) with or without the URS (-629 to -93) were transcriptionally fused to lacZ, each fusion construct was mobilized into R. sphaeroides 2.4.1, and beta-galactosidase activities were determined under the conditions described.

From the data presented in Fig. 2it is obvious that the changes at -68 and -67 had a severalfold effect upon puc operon expression in both the presence and absence of the URS. Further, and most importantly when compared with wild type, the effect was observed regardless of the environmental conditions under which puc operon expression was measured. On the other hand, mutations at -83 and -82 showed little alteration in puc expression in the presence of the URS and little if any change in the absence of the URS.


Figure 2: beta-galactosidase activities associated with transcriptional fusions of the lacZ gene to puc upstream DNA containing either wild type or mutated forms of the putative activator-binding sequence within the DRS. The puc DNA region corresponding to the 19 bp from -84 to -66 is underlinedbelow the restriction map and also shown as a thickline for each puc::lacZ fusion. Mutated bases are denoted with asterisks. The puc::lacZ fusion constructs were present in trans in R. sphaeroides 2.4.1 grown chemoheterotrophically (30% O(2), 1% CO(2), 69% N(2)) or anaerobically in the dark with Me(2)SO. Photoheterotrophic cultures grown at 10 W/m^2 were harvested between 40 and 60 Klett units (1 Klett unit = 0.75 10^7 cells). Deviations for beta-galactosidase measurements are shown in parentheses.



A third mutation, deleting three helical turns, i.e. 32 bp between -87 and -56 as depicted in Fig. 3and designated Deltac, was introduced in the DRS. This deletion encompasses the region of TGGC-N-TCGCA as well as 3 and 10 bp upstream and downstream, respectively. The mutated DRS was transcriptionally fused to lacZ in the presence and absence of the URS and beta-galactosidase activities determined as described in the legend to Fig. 4. The removal of the DNA sequence extending from -87 to -56 resulted in a 3-8-fold decrease in puc operon expression under the conditions employed in both the presence and absence of the URS, when compared with the wild type construction.


Figure 3: A, the puc DNA sequence extending from -93 to +1 illustrating the region mutated through oligonucleotide-directed in vitro mutagenesis. Delta denotes deletion of the area confined by the bracketsabove the sequence. TGT (-52 to -50) and ACA (-37 to -35) were mutated to GCG and CGC, respectively. B, the puc DNA sequence extending from -93 to +1 illustrating the DNA region mutated by selection of regulatory mutations employing the puc::aph fusion. cis mutations at -36 and -11 are shown below the DNA sequence together with the previously described -26 and -12 mutations(12) . Duplication of the 12-bp DNA sequence from -34 to -23 is illustrated above the region of duplication.




Figure 4: beta-galactosidase activities associated with transcriptional fusions of the lacZ gene to the puc upstream DNA containing wild type and various mutations associated with the putative activator(s)-binding region and the regions of dyad symmetry as described in Fig. 3. Each mutation can be identified by noting the precise change to the immediateright of each plasmid designation. The solidlines indicate the presence of either the entire 629-bp upstream sequence or the 92-bp upstream sequence. The puc::lacZ fusion constructs were present in trans in R. sphaeroides 2.4.1 grown chemoheterotrophically (30% O(2), 1% CO(2), 69% N(2)) or anaerobically in the dark with Me(2)SO. Photoheterotrophic cultures at 10 W/m^2 were harvested between 20 and 30 Klett units. Deviations of the beta-galactosidase activities are shown in parentheses. n.d., results not determined here.



One further point is worth noting regarding the level of derepression when puc operon expression is measured in cells growing aerobically and under dark/Me(2)SO conditions. When the URS is present, both the Deltac mutation and the mutations at -68 and -67 still show an approximate 6-fold derepression, which is about 30% of that observed for the corresponding wild type constructions. Thus, despite the overall damaging effect upon puc operon expression, these mutations largely retain the ability of the puc operon to respond to the removal of O(2).

Selection of cis-Acting Mutations Involved in Oxygen and Light Control of puc Transcription

Previously, two cis-acting mutations affecting oxygen regulation of puc operon expression were isolated by exploiting a puc::aph transcriptional fusion(12) . The mutants were selected because of their increased resistance to Km in the presence of O(2). One mutation mapped to -26 (G A) and the second to -12 (A C). Both mutations were silent in the absence of the URS, and both showed increased LacZ expression in the presence of O(2). Other differences were also noted and will be discussed later.

Because these mutations mapped to one of the two regions of dyad symmetry (Fig. 1B) a similar approach (12, 13) to the isolation of additional mutants displaying increased resistance to Km was undertaken using even higher concentrations of Km (40 and 80 µg/ml). Four independent isolates of the same cis-acting mutation at -11 (C T) were isolated at a Km concentration of 40 µg/ml (Fig. 3). At 80 µg/ml Km two additional mutations were isolated: the same at -11 (C T) as above and a duplication of the 12-bp DNA sequence between -34 and -23 (Fig. 3). The 12-bp duplication includes the left half of the proximal region of dyad symmetry plus the gap sequence between the two regions of dyad symmetry (Fig. 3).

Another approach was employed to isolate a different mutation at -36 (C T) (see Fig. 3). In this case increased resistance to Km was selected in cells growing at 100 W/m^2 light intensity. Normally, these cells containing the puc::aph fusion (12, 13) are resistant to up to 2.5 µg/ml Km. At 3 W/m^2 these same cells are resistant to 25 µg/ml Km. Thus, mutants resistant to 100 µg/ml Km at 100 W/m^2 were isolated. Only one cis-acting mutation (at -36) was obtained, and the remainder were shown to be trans-acting mutations, which are not described here.

All of these mutations residing in the DRS were transcriptionally fused to lacZ in the presence or absence of the URS and beta-galactosidase activities measured under various conditions of growth as depicted in Fig. 4. Upon examination of the results, several points are noteworthy. As expected for the mutations at -11 and the duplication of the sequence from -34 to -23, the level of LacZ expression in the presence of O(2) is greater, 5-6 fold, than the corresponding wild type. What was unexpected is that the mutation at - 36 yielded LacZ activity 5-fold greater than wild type in the presence of O(2), although it was selected for in response to altered expression in the presence of high light under anaerobic conditions. However, unlike the former two mutations, the change at -36 did lead to increased expression either under conditions of anaerobic dark or anaerobic light growth, when compared with the wild type. When the DRS was uncoupled from the URS, each of these three mutations resulted in different levels of expression of beta-galactosidase. The mutation at -36 was essentially silent, and the duplication showed decreased expression. However, the mutation at -11 showed increased LacZ expression in the presence or absence of O(2).

Site-directed Mutations of the Distal Region of Dyad Symmetry

Of the five independently isolated mutations involving both the distal and proximal regions of dyad symmetry, four involved the proximal region (see Fig. 1), with only one, namely -36, involving the distal region (see Fig. 1). Using site-directed mutagenesis we created three additional mutations in the distal region of dyad symmetry as follows: Deltad, involving the removal of the entire distal region of dyad symmetry; -52 to -50, involving the change of TGT to GCG; and -37 to -35, involving the change of ACA to CGC (see Fig. 3). These last two mutations were created because of the obvious symmetry in DNA sequence between the distal and proximal regions of dyad, i.e. TGT-N-ACA. Each mutation within the DRS was followed with respect to its effect on lacZ expression in the presence or absence of the URS as described previously (Fig. 4).

In the complete absence of the distal region of dyad symmetry (Deltad) the level of beta-galactosidase in the absence of the URS is near background. Even in the presence of the URS, LacZ levels are low, and there appears to be no increased expression upon removal of O(2).

Also evident is the fact that changes at positions -52 to -50 and -37 to -35 lead to increased LacZ activity in the presence of O(2), and in the case of -52 to -50 increased expression in the absence of O(2) is observed when the URS is present. In the absence of the URS, only the changes at -37 to -35 show increased beta-galactosidase in the presence or absence of O(2), whereas the changes at -52 to -50 are silent when these mutations are compared with the wild type. However, both sets of changes continue to show derepression under an aerobic to anaerobic transition. Thus, it appears that both regions of dyad symmetry play a role in the repression of puc operon expression in the presence of O(2) as well as in derepressed expression of the puc operon in response to the removal of O(2). Also, mutations localized to these regions of dyad symmetry respond differently, depending upon whether or not the DRS and URS are coupled or uncoupled.


DISCUSSION

The puc operon upstream DNA between the XmaIII(-92) and the HaeIII(-57) restriction sites immediately downstream of the overlapping FNR- and IHF-binding sequences was shown previously to play an important role in the expression of the puc operon under both aerobic and photoheterotrophic conditions when the DRS was uncoupled from the URS (12) . For the purposes of this discussion expression of lacZ is considered to be a relative measure of puc operon transcription. The results reported here support further the importance of this DNA sequence centered at approximately -75. We propose that this region serves as a binding site for a positively acting transcriptional regulator(14) . The fact that the presence of this region results in an increased expression of the puc operon whether or not O(2) is present would be anticipated for a generalized transcriptional activator. The mutation at -68 and -67 appears to have a lesser effect in the presence of oxygen than in its absence. This may reflect, as discussed below, the ability of the hypothetical activator to contact RNA polymerase whose promoter binding ability would be strongly affected by the presence of repressor when O(2) is present.

Although the results of this study were focused on the DRS, the question naturally arises as to the role or roles of the URS since there are absolute differences in the expression of the puc operon reported here and elsewhere (12, 13, 14, 15) dependent upon the presence or absence of the URS. One possibility is that a second promoter is present in the URS in addition to a promoter within the DRS. There are several observations arguing against that possibility. First and foremost all attempts to detect puc operon transcripts or 5` ends derived from DNA sequences upstream of the reported unique 5` end (1) have proven unsuccessful despite the method of RNA preparation. Second, the response regulator PrrA having a major effect upon puc operon expression has been interpreted to exert its effect upon puc operon expression through the DRS. Third, the silencing of certain specific mutations (e.g. at -12, -26(12) , -52 to -50, and -36 reported here) when the URS is uncoupled from the DRS is difficult to explain solely on the basis of two promoters. lacZ fusions to either the StuI or XmaIII sites located approximately 225 and 92 bp, respectively, upstream of the start site of transcription, result in only background levels of lacZ expression under various conditions of growth. For example, in the presence of high oxygen the wild type level of LacZ is 163 units of beta-galactosidase/mg of protein, whereas the XmaIII fusion yields 4 units, and the StuI fusion yields 9 units. Similarly, under dark/Me(2)SO conditions the wild type yields 1533 units and the XmaIII and StuI fusions yield 8 and 11 units, respectively. These low levels of beta-galactosidase from the upstream fusions are similar to those observed when lacZ is positioned immediately downstream of an cartridge without any obvious promoter sequences positioned in between. Similar experiments conducted with cells growing under low light intensities have given similar results. It would be necessary to assume that changes in the DRS could affect the upstream promoter, leaving us back where we started. Finally, the exceptionally low levels of residual puc operon expression as well as the absence of any increased expression following the removal of O(2) in the Deltad mutation lying entirely within the DRS supports the existence of a single promoter within the DRS. Therefore, without evidence to the contrary we will continue with the working hypothesis that the interaction of the URS with the DRS is through the mediation of additional trans-acting factors. Our laboratory has defined several potential factors and, as described earlier, an FNR homologue.^2

Where is the promoter within the DRS located? Previously (12, 14) we proposed that the regulatory region of the puc operon possessed the properties of both - and -type promoters(27) . In either instance RNA polymerase can cover DNA coordinates between -50 and +20. In light of the effects of the Deltad deletion on puc operon expression we suggest that the most upstream portion of the promoter has been removed. This interpretation is complicated by the fact that the Deltad deletion has resulted in the removal of less than two helical turns of the DNA, placing the putative activator binding region out of phase with the promoter. However, the Deltac mutation that removes the putative activator binding sequence does not diminish puc operon expression to the extent of the Deltad mutation, and more importantly, the Deltac mutation leaves ± 0(2) control largely intact. Therefore, we propose that the promoter for the puc operon overlaps both the distal and proximal regions of dyad symmetry. This is supported by recent work by Gomelsky and Kaplan. (^3)A similar although not identical case has been observed in the regulatory region of the bchCXYZ operon in R. capsulatus(28) .

Comparison of the 90-bp puc upstream DNA of R. sphaeroides with that of R. capsulatus(25, 26) reveals a high degree of sequence homology from -84 to -66 (putative activator binding regions(s)) and -52 to -28 (distal dyad symmetry and its immediate 7-bp downstream DNA) as well as from -9 to -1. Only one sequence of dyad symmetry was found in the puc upstream DNA from R. capsulatus(25, 26) .

Penfold and Pemberton (29) recently identified a gene, ppsR, which alone is sufficient for photopigment suppression in R. sphaeroides. The ppsR gene encodes a protein with a DNA-binding domain within the COOH-terminal region. These authors suggested that the PpsR protein recognizes the motif TGT-N-ACA, found to reside within the immediate cis-acting sequences of some but not all photopigment genes. We proposed (12) that these same sequences within the DRS of the puc operon are involved in factor binding(17) . In a more direct approach to this hypothesis Gomelsky and Kaplan^3 have determined that the ppsR gene product interacts with these regions of dyad symmetry and that point mutations within these sequences can alter PpsR-mediated repression of puc operon expression.

We now possess genetic evidence that the region of dyad symmetry possessing the TGT-N-ACA motif has repressor binding activity.^3 Because these same regions of dyad symmetry occupy the region -10 to -52, this sequence may also contain the promoter for the puc operon. The net effect of mutations within these sequences could simultaneously reflect alterations in both repressor binding and promoter strength. For example, point mutations having increased puc operon expression in the presence of O(2) may reflect diminished repressor binding without any effect upon the puc promoter. However, we cannot rule out the possibility that other mutations could lead to increased promoter strength without affecting repressor binding. Intermediate alternations between these extremes are more than likely. It is also likely that each region of dyad symmetry is capable of binding repressor, since mutations mapping to both regions lead to increased puc operon expression in the presence of O(2).

If we assume that under anaerobic conditions repressor binding to the region(s) of dyad symmetry is no longer involved in regulating expression of the puc operon, then the mutations at -12, -36, and -52 to -50 may reflect changes in promoter accessibility, the former two directly representing changes in promoter sequence and -52 to -50 indirectly through their effect on the -36 region by virtue of a change in DNA structure. Since these changes are only observed in the presence of the URS, we conclude that the presence of the URS is important for enhanced accessibility of RNA polymerase to the puc operon promoter.

There are two mutations, at -11 and -37 to -35, which in addition to displaying increased puc operon expression in the presence of O(2) do so whether or not the URS is coupled to the DRS. Each of these changes occurs in the proximal arm of both the distal and proximal regions of dyad symmetry. Several interpretations, which are not mutually exclusive, are possible. These mutations might signify asymmetric binding of the repressor to the region(s) of dyad symmetry. Or these sequences may lie within and therefore define the puc operon promoter.

At this point it is worth noting that mutations in the URS sequence located between -197 and -190 led to a 7-fold increase in puc operon expression in the presence of O(2). (^4)This observation is most readily explained if direct interactions, mediated by protein factors, between the URS and DRS can occur. Interactions between the URS and the DRS are further strengthened by the results obtained with the duplication of the region from -34 to -23. In the absence of the URS, RNA polymerase interactions would be destablized and activity would be low, as seen. However, in the presence of the URS and other protein factors proper alignment of those regions might be favored. Any explanations involving transcription initiation from sequences within the URS would still require additional mechanisms for such changes in the URS resulting in silencing of repressor binding within the unaffected DRS.

It is obvious that much remains to be learned regarding the cis-acting regulatory sequences involved in puc operon expression. If we are correct in our assumptions that the regions of dyad symmetry within the DRS serve both to bind repressor (and perhaps other regulatory effectors) and RNA polymerase, then the mutational effects observed here represent the outcome of a subtle interplay between these disparate activities. Therefore, it may be possible to view each of these unique roles through the use of specific genetic backgrounds. For example, in a PpsR minus background it may be possible to undertake an investigation of the puc operon promoter, all else being equal.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM15590 (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biology, SoGang University, C.P.O. Box 1142, Seoul 100-611, Korea.

To whom all correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, P.O. Box 20708, Houston, TX 77225. Tel.: 713-794-1742; Fax: 713-794-1782; skaplan{at}utmmg.med.uth.tmc.edu.

(^1)
The abbreviations used are: bp, base pair(s); URS, upstream regulatory region; DRS, downstream regulatory region; FNR, fumarate nitrate regulator; IHF, integration host factor; W, watts; Km, kanamycin.

(^2)
J. Zeilstra-Ryalls and S. Kaplan, unpublished results.

(^3)
Gomelsky, M., and Kaplan, S.(1995) J. Bacteriol.177, 1634-1637.

(^4)
Y. Q. Wu and S. Kaplan, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. J. Eraso for contributions to this study.


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