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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1991 Academir

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