Transcriptional organization of the Escherichia coli dnaX gene

J. Mol. Biol. (1991) 220, 649-658

Transcriptional

Organization

of the Escherichia coli dnaX Gene

Ann M. Flower? and Charles S. McHenryS Department of Biochemistry, Biophysics and Genetics University of Colorado Health Sciences Center Denver, CO 80262, U.S.A. (Received

16 November

1990; accepted 26 March

1991)

We have determined the transcriptional organization of the Escherichia coli dnaX gene, the structural gene for both the y and z subunits of DNA polymerase III holoenzyme. By S, nuclease protection and primer extension mapping of transcripts encoding the dnaX products, one primary promoter of dnuX has been identified that initiates transcription 37 nucleotides upstream from the first codon. dnuX resides in an operon with two recently sequenced genes, orfl2, encoding an unidentified product, and recR, the structural gene for a protein involved in the recF pathway of recombination. Under conditions of balanced growth, a very small amount of transcription from the upstream apt promoter (~5%) contributes to the expression of z and y, too low for apt to be considered to be on an operon with dnaX. orfl2 and recR are transcribed from an independent promoter as well as from the dnuX promoter, providing a mechanism for orfl2 and recR to be regulated independent of dnaX. Transcription of the dnaX-orfl2-recR operon is terminated upstream from the previously characterized heat shock gene htpG. The dnaX and orfl2-recR promoters, cloned into a promoter detection vector, efficiently direct the expression of the downstream reporter gene, la.cZ. These results extend our knowledge of the genetic and transcriptional organization of this region of the E. coli chromosome. The transcriptional organization has been defined as follows: apt, dnaX-orflZ-recR, htpG. All of these genes are transcribed in the clockwise direction and only dnaX, orfl2 and recR are contained in the dnaX operon. Keywords:

regulation;

RNA mapping; dnaZ; DNA replication;

1. Introduction Replicative DNA synthesis in Escherichia coli is carried out by the complex multisubunit enzyme DNA polymerase III holoenzyme (for a review, see McHenry, 1988). The constituent subunits of holoenzyme, with the exception of b, have been estimated to be at a level of 10 to 20 copies per cell (Kornberg & Gefter, 1972; Otto et al., 1973; McHenry & Kornberg, 1977; Wu et al., 1984). Given these low subunit levels and the essential role of the DNA polymerase III holoenzyme, the synthesis of the subunits must be co-ordinated so that they are present in adequate quantity for assembly into the replicative complex. Co-ordination of gene expression in E. coli is often achieved by the organization of genes with similar functions in a single transcriptional unit, an operon. This mechanism is not used for the co-ordinate t Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, U.S.A. $Author to whom reprint requests should be addressed. 0022%2836/91/15osas-IO

$03.00/0

1acZ

expression of DNA polymerase HI holoenzyme subunits; the structural genes for its subunits are scattered throughout the chromosome. The structural gene for a, the polymerase catalytic subunit, is dnaE, located at four minutes (Gefter et al., 197 1; Wechsler & Gross, 1971; Welch & McHenry, 1982). dnaN, encoding /l, is on an operon with dnaA and recF at 83 minutes (Sakakibara & Mizukami, 1980; Sako & Sakakibara, 1980; Burgers et al., 1981; Armengod & Lambies, 1986; Armengod et al., 1988; Quifiones & Messer, 1988). dnuQ (mutD), encoding E, is transcribed in a divergent manner from rnh at five minutes (Horiuchi et al., 1978, 1981; Echols et al., 1983; Scheuermann et aZ., 1983; DiFrancesco et al., 1984; Nomura et al., 1985). dnaX, directing the synthesis of both the z and y subunits via a frameshifting mechanism, is located at 10.4 minutes (Wickner & Hurwitz, 1976; Henson et al., 1979; Hiibscher & Kornberg, 1980; Kodaira et al., 1983; Mullin et al., 1983; Hawker & McHenry, 1987; Flower & McHenry, 1990; Blinkowa & Walker, 1990; Tsuchihashi & Kornberg, 1990). These genes have all been cloned and the nucleotide sequences

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f’ress Limited

A. M. Flower

650

and C. S. McHenry

determined (Maki et aZ., 1983; Ohmori et al., 1984; Flower & McHenry, 1986; Yin et al., 1986; Tomasiewicz & McHenry, 1987). Genes with similar regulatory requirements may also exist as part of a regulon, a set of independent transcripts that respond to a common regulator (for a review, see Neidhardt, 1987). It is possible that the DNA polymerase III holoenzyme subunit genes are part of a regulon, but a common regulator has not yet been identified. Testing of this hypothesis requires knowledge of the transcriptional organization of the structural genes encoding holoenzyme subunits. The work described in this study was undertaken to determine the transcriptional organization of the dnaX gene to facilitate understanding of how the cell controls expression of the gene products utilized for DNA replication.

2. Materials and Methods (a) Strains and plasmids Total RNA was isolated from E. coli K12 strain MG1655 (A-, F-) (Guyer et al., 1980) that was obtained from B. Bachmann (CGSC strain no. 6300). This strain was considered to be wild-type. Strain D1245 [hsdS20, recA56, A(lac)X74, rpsL20, proA2, ara14, ~~15, mtll, supE44] was a gift from J. Betz of this department and was used as the host for /3-galactosidase operon fusion plasmids. Plasmid pRS415, used for the construction of plasmids containing fusions of E. coli promoters to la&, was obtained from R. Simons (Simons et aZ., 1987). pRS415 contains 4 elements important for this work: (1) a polylinker with EcoRI, SmaI and BamHI restriction sites; (2) 4 tandem copies of the strong transcriptional terminabor Tl from the rrnB operon located immediately 5’ to the polylinker and oriented to block entering transcription; (3) the intact ZucZ gene, including the translation initiation site, but not the transcriptional promoter, positioned 3’ to the polylinker so that inserted promoters will direct the transcript.ion of EucZ; and (4) the wild-type lac Y and 1acA genes. Plasmid pMWZ1101, containing the wild-type apt and dnaX genes and a portion of orfl2 on a 3.1 kbt EcoRI to Pat1 restriction fragment jnucleotides -836 to + 2246 as numbered in Fig. I), was constructed by M. Welch of this laboratory. Plasmid pBJ 1, containing the wild-type apt, dnaX, orfl2, recR and htpG genes and a portion of the adk gene on a 6.0 kb EcoRI to EcoRI restriction fragment ( - 836 to + 5129) was used for construction of probes for mapping the 3’-end of dmX transcripts and was obtained from J. Bardwell. Plasmid pMLB2, used to construct the probe for the ratio S, map of the 5’-ends of the dnuX transcripts was constructed by M. Bradley of this laboratory and contains the wild-type dnaX gene on a 2.35 kb SmaI to Pat1 restriction fragment, (- 101 to +2246) inserted into the expression vector. pKK223 (Pharmacia). Restriction sites used for cloning of promoters into operon fusion vectors and for construction of S, probes are shown in Fig. 1. Numbering of the nucleotide sequence has been changed from that used previously (Flower & McHenry, 1986) so that the 1st nucleotide of the dnaX transcript corresponds to nucleotide + 1 (results t Abbreviations used: kb, lo3 base-pairs; bp, basepair(s); dNTPs, deoxyribonucleoside triphosphates.

RH

S

laprl -

N

X Nh Ss P S/A

Sa

lpjxq~

dnaX

hfpG

I

Nde-1 -

Nde-2 Xma-1

-

Xma-2

l

Ava-1

* l

Sma-1

Figure 1. Restriction map of the dnaX region and probes used for S, analysis. Restriction sites used for cloning and for construction of S, probes are shown. The lines indicate chromosomal portions of S, probes, with the star indicating the labeled end. Abbreviations are: R, EcoRI; H, HindTII; S, SmaI; N, NdeI; X, XmaIII; Nh, NheI; Ss, SspI; P. P&I; A, AmI; and Sa, EaZZ.

presented here). Thus, the Hind111 site that corresponded to nucleotide + 1 in the original numbering is now numbered - 529. (b) Construction

of operon fusion

plasmids

Proposed dnaX promoter Pl (Flower & McHenry, 1986) was cloned by digestion of pMWZ1101 with Hind111 (-529) and SmaI (- lOl), filling in the Hind111 end with phage T4 DNA polymerase, and ligating the 430 bp fragment with pRS415 that had been digested with SmaI. Plasmids containing the insert in both orientations were obtained; the plasmid with the promoter in the forward direction relative to la& was designated pAFP3, and the plasmid with the insert in the inverse orientation was designated pAFP4. Proposed dnaX promoter P2 (Flower & McHenry, 1986; Yin et aZ.. 1986) was cloned from the product of a polymerase chain reaction used to amplify the desired sequences. One oligonucleotide that hybridized to bases - 101 through -82 on the strand used as the transcriptional template contained a 5’-extension with EcoRI and BgZIIrestrictionsites. A 2nd oligonucleotidethat hybridized to bases 81 through 100 on the complementary strand contained a 5’-extension with EcoRI and BumHI restriction sites. These two oligonucleotides were annealed to pMWZ1101 and the polymerase chain reaction was performed as described (Saiki et al.. 1988) except that annealing was performed at 45°C and the extension at 72°C was continued for 2 min/cycle. ,4 portion of the 240 bp product was digested with EcoRI and BamHI and ligated into pRS415 that had been cut with EcoRI and RamHI, giving rise to pAFP5, containing the promoter in the forward direction relative to la&. To obtain pAFP6 with the promoter in the reverse orientation, a portion of the 240 bp product of the polymerase chain reaction was digested with EcoRI and BgEII and cloned into pRS415 that had been cleaved with EcoRI and BamHI. Promoter fusions containing the orfl2-recR promoter were obtained by digesting pMWZllO1 with NheI (1574) and SspI (1945), filling in the NheI ends with TP DNA polymerase, and ligating into pRS415 that had been

Mapping

of E. coli dnaX Transcripts

digested with SmaI. Clones with the insert in both orientations were obtained; the clone containing the promoter in the forward direction was designated pAFP7, and that in the inverted direction, pAFP8. (c) Enzymes and reagents Restriction enzymes, T4 DNA polymerase, T4 DNA !igase, and T4 polynucleotide kinase were obtained from New England Biolabs and were used according to manufacturer’s instructions. Avian myeloblastosis virus (AMV) reverse transcriptase was purchased from Life Sciences, Inc. Nuclease S, was from Boehringer-Mannheim Biochemicals. Penicillinase (Sigma) and /I-galactosidase (Boehringer-Mannheim) were used as standards for and o-nitrophenyl-fi-nenzyme assays. Cephaloridine galactoside used as subst,rates were obtained from Sigma. (d) Labeling

and purijcation

of probes

8, probes were designed so that each contained plasmid DNA extending beyond the chromosomal DNA at the unlabeled end, allowing the differentiation of products that were full-length due to incomplete digestion of the probe from products that were full-length due to the mRNA extending past the end of the probe. Radioactive labeling of S, probes and of oligonucleotides was performed by standard procedures (Maniatis et al., 1982). Phosphorylation of 5’-ends was performed using polynucleotide kinase in the presence of a 2-fold molar excess of [Y-~~P]ATP (ICN Biomedicals, Inc.) to produce 5’-endlabeled probes. Recessed 3’.ends generated by restriction endonuclease digestion were filled-in with T4 DNA polymerase in the presence of the necessary dNTPs, one of which was labeled with 32P at the u position (ICN Biomedicals. Inc.) and was present in molar excess to produce 3’.end-labeled probes. Probe Nde-1 (Fig. I) was constructed by digestion of pMWZ1lOl with NdeJ that, cuts at nucleotide 423 of dnaX and at 2296 of pBR322. The 3555 bp fragment containing the amino-terminal dnaX sequences was puritied and 5’-end-labeled. The probe contained 1259 bp of apt and dn~X sequences and 2296 bp of pBR322 sequence. Probe Nde-2 (Fig. 1) was constructed by digesting pMLB2 with ,VdeT. The enzyme digests the dnaX portion of pMLB2 at the same positions as pMWZl101; however, the plasmid does not, contain the apt sequences from EcoRl to BmaT. therefore the probe contains only 524 bp of dnaX sequences. Probe Xma-1 (Fig. I) was constructed by digestion of pMWZ1101 with XmaIII that digests the dnaX sequences at nucleotide 1357 and the pBR322 sequences at nucleotide 938. The 3565 bp probe was purified and 3’-endlabeled and contained 891 bp of dnaX sequences, including the dnaX-orfl2 intercistronic region, and 2674 bp of pBR322 sequences, Probe Xma-2 (Fig. 1) was constructed by digestion of pBJl with XmaIII. pBJ1 is cut by XmaIII at nucleotide 1357 of dnuX and at 938 of pBR322. However, there is an additional 2.9 kb of chromosomal DNA downstream from dnaX in pBJ1, and the chromosomal insert was in the opposite orientation with respect to pBR322 as in pMWZ1101. The probe was therefore 4676 bp in length, containing 3776 bp of rhromosomal DNA and 900 bp of pBR322 DNA. Probe Ava-I (Fig. 1) was constructed by digestion of pBJ1 with AvaI. AvaI digests within orfl2 at nucleotide 2966 and within the pBR322 sequences at position 1424.

651

The resultant probe was therefore 4423 bp, containing 2999 bp of chromosomal DNA and 1424 bp of plasmid DNA. Probe Sma-1 (Fig. 1) was constructed by digestion of pBJ1 with SmaI that cuts within orfl2 at nucleotide 2968 and also upstream from dnuX (-101). The probe was 3061 bp in length and consisted of only chromosomal sequences. (e) RNA

techniques

Purification of total E. coli RNA from exponentially growing cells was performed by extraction with hot phenol and precipitation with sodium acetate (Sharma et al., 1986) except that NaF was omitted from the extraction buffer. Primer extension reactions were performed as described (Singh et al., 1985) except that annealing was performed by incubating for 3 min at 95°C. followed by cooling slowly to room temperature. Endonuclease S, digestions were performed essentially as described (Berk & Sharp, 1978), except that hybrrdizations were carried out by an initial denaturation for 3 min at 95°C followed by incubation at 53°C overnight, and the S, digestion was performed at 37°C for 30 min. Primer extension and S, digestion products were analyzed by electrophoresis on denaturing polyacrylamide gels (8 M-urea) as described by Maniatis et a,Z. (1982). (f) Enzyme assays Lysis of exponentially growing cells was carried out by lysozyme treatment, freeze-thaw and sonication as described (Lupski et al., 1984). &galactosidase and /I-lactamase assays were performed as described (Miller, 1972; Lupski et aE., 1984)‘except that the assays were performed by continuous monitoring of the change in absorbance using the kinetics software for t.he Hewlett Packard 84518 spectrophotometer. Because the absorbance was monitored continuously, the fl-galactosidase assays remained at pH 7.0 and were not quenched by the addition of NaCO,. Because of the difference in pH, the extinction coefficient was 067 times the value normally obtained. The fi-galactosidase values presented here have been corrected for this altered extinction coefficient by multiplying the obtained value by 1.5. The /%galactosidase activity is expressed in Miller units. Protein det,erminations were made by the BioRad Bradford assay using a y-globulin standard.

3. Results (a) Detection of $-term&i

of dnaX

mRlVAs

On the basis of homology to consensus promoter sequences, t,wo potential transcriptional promoters (Flower & for the dnuX gene were identified McHenry, 1986). The site designated Pl is located from nucleotides -203 to - 176; the second site, P2, is located at nucleotides -34 to - 6. To determine whether these proposed promoters were functional, SI protection experiments were performed using the 3555 bp probe Nde-1 that was 5’.endlabeled within dnaX and extended past both proposed promoters PI and P2. Two products (424 bases and 1170 bases) resulted from this S, reaction (Fig. Z(a), lane 1). The very faint longer product corresponded to a transcript initiating at the previously characterized apt promoter (P,,,) (Hershey & Taylor, 1986), and the shorter product

652

A. M. Flower and C. S. McHenry

1

C

T

1018 952

(b) Figure 2. Mapping of the 5’-end of dnaX. (a) S, mapping of dnaX with probe Nde-1. Lane 1, S, reaction with total E. coli RNA resulted in products of 1170 and 424 bases, marked by arrows on the left; lane 2, Nde-I probe digested with EcoRI to result in a 952 bp fragment; lane 3, Pu’de-1 probe alone digested with S,; lane M, size markers with sizes indicated on the right in bp. (b) Primer extension with an oligonucleotide complementary to bases 83 through 99 within dnaX. Lane 1, primer extension reaction with total E. coli RNA, with arrows marking the 5’ ends of the sequence; lanes C and T, sequencing reactions containing ddCTP and ddTTP, respectively, primed with the same oligonucleotide.

mapped immediately upstream from dnaX in the vicinity of P2. The initiation point of the transcript more precisely by mapping to P2 was identified primer extension using oligonucleotide primer p290 that hybridizes to dnaX mRNA from bases 83 through 99. Electrophoretic analysis of the primer extension product alongside a sequencing ladder generated by extension from the same primer allowed us to identify the 5’-end to single nucleotide resolution (Fig. 2(b), lane 1). Primer extension results in a doublet that maps to the C at position - 1 and the T at position + 1. Many polymerases, including AMV reverse transcriptase used in this experiment, add an additional non-templatedirected base to the product of primer extension reactions (Clark, 1988). Therefore, we regarded the shorter product mapping to the T as the authentic 5’-end. No evidence for transcription initiating at proposed promoter Pl under conditions of balanced

growth has been obtained. A transcript corresponding to PI would have resulted in an S, protected fragment of about 592 bases (Fig. 2(a), lane 1 ), and a primer extended product of 268 bases (Fig. 2(b), lane 1). Since P2 was the only detected promoter under these conditions it is designated P dnaX’ (b) Origin

of the

S-end of the dnaX

transcript

Hershey & Taylor (1986) defined the transcription map of apt as initiating upstream from apt (-746, using our numbering system) and terminating in the apt-dnaX intercistronic region. One of the two was detected 3’.termini of the apt transcript mapped to position + 1. As a portion of the dnuX transcription arises from the apt promoter, it was possible that the shorter transcript apparently directed by P&,=x might have arisen by processing of

of E. coli dnaX Transcripts

Mapping

653

Table 1 b-Galactosidase activity of promoter fusions Specific activity? Promoter

Plasmid

NOW

PRY415 pAFP3 pAFP4 pAFP.5 pAFP6 pAFP7 pAFP8

Pl (fwd) Pl(bwd) Pmdfwd) P,n,x(bW Por~,~(fwd) I’or,,,(bwd)

t Miller units/mg total cell protein

<25 400 150 4100 300 4600 500 (Miller,

1972). fwd, forward;

a long transcript rather than by initiation of transcription. To address this issue, we cloned a segment of DNA containing Pdnax but lacking Part (bases - 101 to + 100) into a promoter detection vector pRS415). Pdnsx directed the expression of significant levels of fl-galactosidase (Table 1) and was considered an independently functional promoter. (c) Contribution of apt transcription dnaX expression

to

To determine the extent that readthrough transcription from the apt transcript affects the expression of dnaX, we measured the ratios of RNAs originating from P,,, to those originating from PdnaX.This was accomplished by the use of the 2525 bp Nde-2 S, probe that was labeled within the dnaX gene and extended 100 bases upstream from PdnaX to the &ma1 site. Beyond the SmaI site the probe contained plasmid DNA that was not complementary to the dnaX mRNA. Therefore, three bands were expected, undigested probe (2525 bases), probe annealed to the transcript initiating at P,,, (524 bases) and probe annealed to the transcript initiating at PdnaX (424 bases) (Fig. 3, lane 1). The ratio of the products indicated the ratio in vivo of the two mRNAs. Since the sizes of the two Si products were similar, preferential degradation of a long hybrid was minimized (H. G. Tomasiewicz & C. S. McHenry, unpublished results). Densitometry of autoradiograms from multiple exposures indicated that 97yo of the transcription entering dnaX originates at PdnaX; only 3% originates from sites further upstream. (d) Determination

qf the 3’-end

/I-Lactamase

j-Galactosidase

of the dnaX transcript

To detect the St-end of the dnaX transcript, we used a probe that was 3’-end-labeled at the XmaIII site (1355) and extended to the P&I site within orfl.2 (2246) (S, probe Xma-1). The only product of this reaction resulted from protection of the fulllength probe (891 bases), indicating that no transcription termination occurred within the dnaXorfl2 intercistronic region. A second probe was constructed from pBJ1 that was labeled at the

1500 1600 1600 1850 2000 1250 1200

B-Galactosidasr/P-lartamase < 0.02 0% 010 2.20 0.15 3.70 0.40

bwd. backward.

XmaIII site (1355) and extended through orfl2, recR, htpG and into adk to the EcoRI site (5965) (Xma-2). The product of this reaction was greater than 1509 bases, indicating that the 3’-end of the dnaX transcript was further than 1500 bases downstream from the XmuIII site within dnaX. To map the end more precisely, we used a 4423 bp probe that was labeled at the AvaI site within oTfl2 and extended through recR and htpG to the EcoRI site within adk (Ava-1). The product of this S, digestion was 500 (+5) bases in length (Fig. 4, lane 1). From

424 -396

Figure 3. Determination of the contribution of I’,,, to generation of dnuX transcripts. Lane 1. S, reaction with total E. coli RNA using probe Nde-2 resulted in produets of 424 and 524 bases (full-length probe resulted from incomplete digestion of excess probe); lane 2, Pr’de-2probe alone digested with S,: lane 3. Nde-2 probe undigested; and lane M, size markers in bp.

A. M. Flower

654

and C. S. McHenry

UC A A G-C E g:z

1018

g:g * A-U * UUUAAGCAAA%:&iAUUAUU Figure 5. Predicted stem-and-loop structure near termination of dnaX mRNA. Asterisks indicate the htp(^: promoter and the underlined bases indicate the approximate t,ermination of dnaX transcription.

of - 196 kcal/mol using the method of Zuker & Stiegler (1981) and may cause stalling of the RNA polymerase and termination (for reviews, see Brendel et al., 1986; Yager & von Hippel, 1987). (e) Detection of the Zend

500

398

Figure 4. S, mapping of the 3’.terminus of dnaX using probe Ava-1. Lane M, size marker in bp; lane 1, S, reaction with total E. coli RNA and probe Ava-1 resulted in a product of 500 bases indicated by the arrow on the right.

the sequence of the htpG gene (Bardwell & Craig, 1987), we determined that the 3’-end of the dnaXorfl2-recR transcript is located approximately 75 bp upstream from the initiation point of htpG translation within the recR-htpG intercistronic region. This position overlaps the -35 region of the heat shock promoter directing transcription of htpG (Bardwell & Craig, 1987; Cowing et al., 1985). There is no sequence that resembles the canonical rhoindependent termination site (G + C-rich hairpin followed by a string of T residues); however, a sequence immediately preceding the stop site was predicted to form a stable stem-and-loop structure (Fig. 5). This structure has a calculated free energy

of the orfl2-recR

transcript

On the basis of sequence analysis. we proposed that another potential promoter existed in the carboxy-terminal portion of dnaX (positions 1787 through 1815) that could initiate transcription for the downstream open reading frame (Flower & 1986). We searched for functional McHenry, promoters within the 3’.end of dnaX with the 3061 bp S, probe Sma-1 that was 5’-end-labeled at the SmaI site within recR. We observed several 5’-ends, one mapping to the expected start site for the promoter (P,,,,,,), and several smaller products (Fig. 6(a), lane 1). Primer extension with oligonucleotide p34 (hybridized to nucleotides 2055 through 2071) also revealed several products, mapping to the same locations (Fig. 6(b), lane 1). To determine whether these sites were all authentic 5’.ends, we used primer extension to map the 5’-end of the message produced by the orfl2-recR promoter cloned into the fusion vector, pRS415 (pAFP7), by using an oligonucleotide primer that hybridized to bases 61 to 80 of the pRS415 1acZ gene, 60 nucleotides downstream from the fusion point. The only major product observed corresponded to the proposed promoter (PorflZ) (Fig. 6(c), lane 1). (f) Cloning of promoters

in operon fusion

vectors

Promoters Pl, PdnaX, and PorflZ were cloned into /3-galactosidase operon fusion vectors to assess their ability to function as independent promoters. The Hind111 to SmaI fragment containing Pl demonstrated no significant measurable promoter activity (<500 units/mg total cell protein), consistent with our finding that no mRNA 5’-termini mapped to this site. Fusions containing the fragment with P dnaX expressed 4100 units per milligram activity,

Mapping

1

2

65.5

of E. cob dnaX Transcripts

M

M

1

-1018

220 201

690

- 517

-

160

-506

-

-396

(a)

1

cc> M

-

245

298

Figure 6. Mapping of the 5’-end of orfl%-recH. (a) S, mapping with probe Sma-1 . Lane I, S, reaction with total E. coli RNA, with the longest product of 690 bases marked by the arrow on the left; lane P, probe Sma-I digested with S,; lane M. size marker in hp. wit,h sizes indicated to the right. (b) Primer extension with an oligonucleotide complementary to bases 6055 through 2071 within orfl2. Lane 1, primer extension reaction yielding a major product of 245 bases, marked by the arrow on the left; lane M. size marker in bp, with sizes indicated to the right. (c) Primer extension on mRSA resulting from a Porf12 -la& fusion using an oligonucleotide that hybridizes to bases 61 through 80 wit’hin TacZ. Lane 11. size marker in bp, with sizes indicated to the left: lane 1. primer extension reaction yielding a product of I60 bases. marked by the arrow on t.he right.

-

(b)

-

220

-

201

slightly less than the activity demonstrated by the XheI to SspT fragment containing Porfl 2 (4600 units/ mg). The observed activity of each of the promoters was normalized for plasmid copy number by assaying P-lactamase activity. After applying this correction we found that PorflZ has 1.7 times as much activity as Pdnax. Ail of these fragments were also cloned in the inverse orientation for two reasons. First, background activity in these fusions has been shown to be partially dependent on the length of the DNA fragment cloned (Simons et nl.. 1987). The fragment in the inverted orientation helps to control for this background activity. Second. there are examnles of Penes in E. coli that

A. M. Flower

656

and C. S. MeHenry

are regulated by the production of antisense RNA (Simons & Kleckner, 1983, 1988; Mizuno et al., 1984). Cloning of these regions in the inverse orientation may permit the detection of antisense promoters on these DNA fragments. None of these fragments demonstrated significant activity in the inverted orientation ( < 500 units/mg).

RH

R

Sa

P

Papt *

PdnaX Porl12 -

4. Discussion We have determined the transcriptional organization of the E. coli dnaX gene, that encodes the z and y subunits of DNA polymerase III holoenzyme, and have completed the transcriptional mapping of the entire region of 194 minutes (Fig. 7). This region contains several genes encoding proteins involved in DNA metabolism: apt, encoding adenine phosphoribosyltransferase; dnuX, encoding the z and y subunits of DNA polymerase III holoenzyme; and recR, a recombination gene. In addition, orfl2, with an unknown function, and htpG, a heat shock gene, map to this region. We have shown that dnaX is expressed as an operon with two downstream open reading frames (orfl2 and recR). Transcription of the operon is directed from Pdnax starting 37 nucleotides in front of the initiating AUG codon of dnaX. It had previously been demonstrated that apt is contained on a single transcriptional unit (Hershey & Taylor, 1986), and we determined that the chromosomal expression of this gene is the same as was shown for the gene carried on a plasmid (data not shown). Although we detect some transcription from the upstream apt promoter extending into the dnaX gene, the contribution of this transcript to the total expression of dnaX is extremely low (3%). Therefore, we do not consider apt to be part of the dnaX operon . orfl2 and recR were expressed from an independent promoter (PorflZ) as well as from the dnaX promoter (Pdnsx). The activity of PorflZ is slightly higher than that of Pdnax in the /3-galactosidase expression assay, indicating that orfl2-recR transcripts may be slightly more abundant than dnaXorfl2-recR transcripts. PorflZ resides entirely within dnaX coding sequences, possibly providing a mechanism for additional regulation. A similar situation, with the promoters for a downstream gene residing entirely within the upstream gene, exists for the dnaN and recF genes. This has been postulated to be an important feature for disco-ordinate regulation as the transcriptional process may interfere with translation (Armengod & Lambies, 1986). It has been suggested that orfl2 and recR are translationally coupled (Mahdi & Lloyd, 1989), perhaps providing another level of regulation for these two genes. It had previously been demonstrated that htpG is expressed as a single heat shock-inducible cistron with no detectable expression from dnaX-orfl2-recR (Cowing et al., 1985). We also did not detect transcription continuing into htpG. All detectable tranwithin the recR-htpG scripts were terminated

‘htpG

*

Figure 7. Summary of dnaX transcript mapping. Mapping of the a@ transcript was performed by Hershey & Taylor (1986), and of the h&G transcript by Cowing et al. (1985). Mapping of dnaX, orfl.2 and recR mRNAs are the results of this work. For abbreviations of restriction enzymes, see the legend to Fig. 1.

intercistronic region immediately following a predicted stable stem-and-loop structure (Fig. 5). The genetic organization of this region is very similar to the comparable region of the Bacillus subtilis genome, in which five open reading frames have been postulated to form an operon. These reading frames exist in the order dnaX-orfl07-recMThe dnaX, orflO7, and recM genes orf74orf87. exhibit strong sequence similarity to the E. coli dnaX, orfl2 and recR genes, respectively (J. C. Alonso, K. Shirahige & N. Ogasawara, unpublished results). It has not been demonstrated that these genes reside on an operon; however, the location of proposed promoters is similar to that in the E. coli operon. It also has not been determined whether the B. subtilis dnaX gene also encodes two protein products via a frameshifting mechanism, as does the E. coli dnaX gene (Flower & McHenry, 1990; Blinkowa & Walker, 1990; Tsuchihashi & Kornberg, 1990). The subunits of DNA polymerase III holoenzyme are present in the cell at low levels. The high level of activity of the dnaX promoter, however, is not compatible with a poorly expressed transcript. Pdnax expresses one-fifth of the amount of P-galactosidase (4100 units) as the strong fully induced lacUV5 promoter (22,000 units) observed by Simons et al. (1987). The low level of r and y synthesis is likely controlled through inefficient translational initiation. We predicted previously that the translation initiation signals would be weak (Flower & McHenry, 1986); this is supported by the observation that strong overproduction of r and y can be achieved only by replacement of the native translation initiation signals with synthetic ones (Tsuchihashi & Kornberg, 1989; McHenry et al., 1989). It has previously been suggested that translation initiation is an important regulatory step in the synthesis of many prokaryotic proteins (for reviews, see Gold & Stormo, 1987; Andersson & Kurland, 1990; McCarthy & Gualerzi, 1990) and has been postulated to be the basis of maintaining a low but constant level of expression of the 1acZ gene product.

Mapping

of E. coli dnaX Transcripts

Translational control of expression has already been demonstrated to be important in regulating the expression of the two protein products of dnaX, T and y. The y subunit is produced as the result of a ribosomal frameshift. Although this frameshift has been shown to occur with close to 50% efficiency when the dnaX gene is contained on a plasmid (Flower & McHenry, 1990), the level of frameshifting from the chromosomal dnaX has not been determined, nor is it known whether the frequency of frameshifting is regulated or is constant. Understanding the interplay between potential transcription and translational signals in regulating the synthesis of these critical replication proteins will provide exciting avenues for future research.

We are grateful to ,J. Mills for oligonucleotide synthesis, and H. Tomasiewicz for helpful discussions. This work was supported by grant ROI GM 36255 from the National Tnstit,utes of General Medical Sciences.

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by R. Schleif