Regulation of the operon encoding ribonucleotide reductase in ...

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Department of Biochemistry, University of Minnesota, St. Paul, MN 55108,. USA ... mRNA (nrd mRNA) levels in thymine-starved cells (Hanke and. Fuchs, 1983). .... in the following experiments galK values were normalized to ac- count for thisĀ ...
The EMBO Journal vol.5 no.5 pp. 1077 - 1085, 1986

Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control

Christopher K.Tuggle and James A.Fuchs Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, USA Communicated by A.Munch-Petersen

The ribonucleotide reductase genes (nrd) are induced by thymine starvation. Deletion analysis of the sequences upstream of the cloned nrd genes was used to identify several regulatory regions. The start of transcription (nrdP) was mapped 110 bp upstream of nrdA, the first structural gene. A site required for positive regulation of nrd was mapped 135 bp upstream of nrdP in a region with two direct repeat sequences as well as potential secondary structure. Two other sites (one upstream of nrdP, the other downstream) were identified as sequences whose deletion markedly increase expression. These latter sites show sequence homology and probably interact since the effects of their individual deletion are not additive when combined. Key words: ribonucleotide reductase/gene fusion/nrdP/regulatory sites Introduction Ribonucleotide diphosphate reductase (RDP reductase) is an essential enzyme for all dividing cells, catalyzing the first reaction unique to de novo synthesis of deoxyribonucleotides (Thelander and Reichard, 1979). The Escherichia coli enzyme, which serves as a model for most organisms, consists of two nonidentical subunits. The activity of the holoenzyme is intricately controlled by allosteric effectors binding at sites localized to the large subunit, B1. The primary structure of B 1 and that of the analogous subunits in mouse and herpes virus have been recently compared and shown to share several regions of striking homology (Caras et al., 1985). The smaller subunit, B2, contains a unique stable free radical localized to a tyrosine ring which is required for enzymatic activity (Thelander and Reichard, 1979). A conserved tyrosine found in the B2 subunit of several organisms (E. coli, clam, herpes and Epstein-Barr viruses) has been postulated as the free radical tyrosine residue of the active site (Sjoberg et al., 1985). The synthesis of the two subunits of RDP reductase in E. coli is coordinately regulated (Fuchs, 1977). Synthesis of RDP reductase increases when DNA synthesis is inhibited by thymine starvation, chemical inhibition of DNA elongation or a shift to on-permissive temperature in dna mutants (Filpula and Fuchs, 1977, 1978). Although these conditions also cause induction of another set of enzymes (the 'SOS' response), control of RDP reductase is different since it is independent of recA, recB, recC or lexA gene products (Filpula and Fuchs, 1977). This increased RDP reductase activity is due to increased RDP reductase mRNA (nrd mRNA) levels in thymine-starved cells (Hanke and Fuchs, 1983). Since the half-life of nrd mRNA was unchanged in these cells (Hanke and Fuchs, 1983), it was proposed that induction was due to an increased transcriptional rate of the nrd IRL Press Limited, Oxford, England

operon. The increased rate of transcription was found to be dependent on concomitant protein synthesis: nrd mRNA induction upon thymine starvation is eliminated by simultaneous addition of chloramphenicol or removal of essential amino acids (Hanke and Fuchs, 1984). The nrd operon is contained within a 12 000 bp PstI fragment. This fragment has been cloned into the plasmid vector pBR322 (Platz and Sjoberg, 1980) and the nucleotide sequence of the entire nrd operon, as well as several kilobases of flanking sequences, has been reported (Carlson et al., 1984). To identify both the transcriptional start site and the region of DNA responsible for regulation of nrd mRNA synthesis, restriction fragments covering the 1641-bp 5' to the start of the open reading frame for B I were cloned into several pKO vectors. These plasmids contain a promoterless galactokinase (galK) gene (McKenney et al., 1981). DNA fragments inserted in these plasmids can be assayed for promoter strength and response to in vivo regulatory signals by measuring galK activity in a galK mutant strain. The initiation site for nrd transcription (nrdP) was mapped first to a 1 19-bp fragment, and then precisely identified using SI digestion of DNA/RNA hybrids. The minimal region required for a correct regulatory response to thymine starvation was identified by deletion analysis of the shortest inducible nrd restriction fragment. Two regions were identified by deletion analysis that appear to be involved in repression of nrd transcription. Effects of addition of multiple copies of nrd on the expression of the chromosomal copy of nrd were also investigated. The implications of these results are discussed and a model for the regulation of synthesis of RDP reductase is presented.

Results Restriction mapping ofnrdP and the region required for induction of nrd: expression of nrd is independent of copy number Induction of RDP reductase encoded by nrd carried on pPS2, a pBR322 derivative, by thymine starvation indicated that the nrd genes had not been induced to the level observed for a single copy chromosomal gene (data not shown), possibly because the assay used depends on several cellular proteins that may be limiting at these high levels of RDP reductase. Platz and Sjoberg (1984) have recently shown this to be the case. To circumvent this problem and to obtain an easier as well as more accurate assay of nrd regulation, gene fusions were utilized. A promoterless galactokinase (galK) gene with upstream translational stop codons in all three reading frames has been inserted into a pBR322 derivative and named pKO0 (McKenney et al., 1981, Figure 1). Two pKO0 derivatives, pKO9 (Adams and Hatfield, 1984) and pKOTWl (T.Warner, unpublished) were utilized in this work to clone several restriction fragments located near nrd to map the sites of transcriptional initiation and regulation. The size of nrd DNA fragments cloned varied from 1641 (KpnI - EcoRI) to 119 (Mnll - EcoRI) bp (Figure 1). The EcoRI site was chosen as the 3' border in these fragments since the site maps only 8 bp from the presumed ribosome binding site for nrdA (Carlson et al., 1984) and thus the nrd promoter (nrdP) was ex1077

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Fig. 1. Construction of nrd-galK transcriptional fusions. The top line is a BamHI restriction fragment of nrd DNA from pPS2 (Platz and Sjoberg, 1981). Several restriction subfragments of the region 5' to nrdA were cloned into pKO9 or pKOTWl (see Materials and methods for plasmid constructions) to measure promoter activity and response to in vivo regulatory signals. The bottom line shows the details of the pKO assay system. MCS, multiple cloning sites; V V V, transcriptional stop codons in all three reading frames; CP, constant polarity region.

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Time (min) Fig. 3. Use of nrd- GalK fusions to map nrdP and the regulatory region responsive to thymine starvation. Cultures of N 110 containing various plasmids (see Figure 1) were starved for thymine and samples taken at 60 min intervals following thymine removal for galK assays. GalK activity was expressed as nmol galactose phosphorylated per min per fmol plasmid (Adams and Hatfield, 1984) to correct for variations in plasmid copy number. GalK activity in Nl 10 containing pKG1800 increased slightly with the new protocol and all data points for the nrd-galK fusions were normalized to N1 10/pKGl800 (data not shown). (0), pCKT2; (0), pCKT8; (A), pCKT25; (A), pCKT36; (O), pCKTl22.

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Fig. 2. GalK and RDP reductase assays in cells containing single and multiple copies of nrd. Cultures of N 110 containing pKG1800 (0) or pCKT2 (A) were starved for thymine and samples taken at 30 min intervals after thymine removal for RDP reductase (filled symbols) and galK (closed symbols) assays. GalK assays were performed and GalK units (in nmol galactose phosphorylated per min per ml of cells at OD650 = 1.0) were measured (McKenney et al., 1981). RDP reductase activity (in nmol dUMP produced per min per mg protein) was measured by the procedure of Filpula and Fuchs (1977).

pected to map 5' to the EcoRI site. The largest fragment, Kpn-EcoRl, was cloned upstream of galK in this system (pCKT2) and tested for the ability to regulate correctly galK activity after thymine starvation of the host bacterium. Figure 2 shows the results of thymine starvation of a strain carrying either 1078

pCKT2 or pKG1 800, a control plasmid with galK controlled by galP (McKenney et al., 1981). GalK and RDP reductase activities increase in parallel in pCKT2, showing that the nrd restriction fragment regulates galK activity in this system in the same manner as the chromosomal nrd operon is regulated. The kinetics of expression of the chromosomal nrd operon in a strain with either plasmid do not differ, indicating that multiple copies of the plasmid-borne nrd fragment do not affect expression of the chrQmosomal copy of nrd. There is a small increase in galK activity during thymine starvation in cells containing pKG1800 and in the following experiments galK values were normalized to account for this small non-specific increase in galK activity during thymine starvation. To eliminate possible differences in plasmid copy number of different constructs, galK activity was expressed per amount of plasmid (Figure 3 legend). Additional restriction fragments of the nrd operon were cloned upstream of galK in this system to map nrdP and the region required for induction. As shown in Figure 3, all plasmids except pCKT122 show a low level of galK expression at time 0 of thymine starvation (the galK activity seen for pCKT122 was not significantly above galK activity seen for plasmid without the insert). Therefore, nrdP was tentatively mapped within the 19-bp MnlI EcoRI fragment present in all constructs except pCKT122 (Figure 1). However, only the KpnI EcoRl (pCKT2) and the Sau3A-EcoRI (pCKT8) fragments appear to contain information required for induction of galK activity upon thymine starvation. Since the HpaII EcoRl fragment (pCKT25) does not respond to thymine starvation, a required section of the regulatory must be located in the 132 bp between the Sau3A and HpaI sites present in pCKT8 but not pCKT25. Since a plasmid without this 132-bp region shows a low level of expression that cannot be -

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Fig. 4. Use of SI nuclease to map the transcriptional start of nrd. (a) mRNA from CR34 with or without pPS2 was isolated (Summers, 1970) either before or after 180 min of thymine starvation. The DNA probe (a Sall -EcoRI restriction fragment from pCKT8 radiolabelled at the EcoRI site with polynucleotide kinase) and either 5 or 50 jig of mRNA were hybridized (Berk and Sharp, 1977), digested with SI nuclease (Weaver and Weismann, 1979) and run on a DNA sequencing gel next to a set of dideoxy sequencing reactions. Lanes: A-D, unstarved mRNA; A: 5 tg CR34, B: 50 Ag CR34, C: 5 yg CR34/pPS2, D: 50 Ag CR34/pPS2, E: DNA probe without mRNA or S1 nuclease treatment, F: DNA probe alone, treated with S1 nuclease, G-J, starved mRNA; G: 5 ug CR34, H: 50 Ag CR34, I: 50 Ag CR34/pPS2, J: 5 Ag CR34/pPS2. An autoradiogram of the dried gel shows six protected fragments of 84-89 nucleotides in lanes D (barely visible) and G-J. (b) DNA sequence of nrdP showing initiation sites as well as the -10 and -35 region, compared with prokaryotic promoter consensus sequence (Hawley and McClure, 1983). Capital letters in nrd designate identity to the consensus sequence, where capital letters indicate degree of conservation (von Hippel et al., 1984). +1 indicates site of consensus transriptional initiation, and slashes above the nrd sequence correspond to the six protected fragments indicated in A. Arrows indicate the position of an inverted repeat sequence (Carlson et al., 1984).

induced by thymine starvation, this region must contain a site for positive regulation. Induction of RDP reductase encoded by the chromosomal genes in cells containing these constructions was tested by measuring RDP reductase activity following thymine starvation and was found not to differ from a control strain (data not shown). Mapping of nrd transcriptional initiation site Studies with the nrd-galK fusion plasmids indicated that the

transcriptional initiation site of the nrd operon lies within the 1 19-bp Mnll - EcoRI fragment (Figures 1 and 3). To define precisely the 5' end of nrd mRNA, a 350-bp Sall - EcoRI restriction fragment from pCKT8 (the Sail site maps upstream of the BamHI site in pKO9) was labelled at the EcoRI site with T4 polynucleotide kinase and hybridized (Berk and Sharp, 1977) to mRNA isolated from exponentially growing or thymine-starved cells with or without pPS2. The DNA-DNA hybrids were treated with SI nuclease (Weaver and Weissmann, 1979) and the SI-resistant products were electrophoresed next to a DNA sequencing ladder as a size marker. An autoradiogram of the resulting gel is shown in Figure 4a. The 5' end of nrd mRNA maps at nucleotides -84 to -89 relative to the EcoRI site terminal T residue. Multiple ends are observed for both chromosomal and plasmid encoded nrdP in thymine-starved cells (lanes g -j), as well as in uninduced plasmid-containing cells (lane d, barely visible). Figure 4b shows the DNA sequence at nrdP, which is upstream of the region suggested to be nrdP (Carlson et al., 1984) solely on the basis of sequence homology to the consensus promoter sequence. The DNA sequences at the -35 and -10 regions of nrdP shows strong agreement with the consensus promoter sequence (Hawley and McClure, 1983). Such agreement with the consensus sequence usually correlates well with a high promoter strength (Raibaud and Schwartz, 1984), but nrdP is relatively weak (compare galK expression in pCKT2 versus pKG1800 at time 0 in Figure 2). Secondary structure involving both the -35 and -10 regions of nrdP (Carlson et al., 1984) (Figure 4b) could lower the in vivo strength of nrdP, or additional regulatory mechanisms may be functioning to repress transcription at nrdP. Deletion mapping of nrd region required for induction To define the region of DNA involved in regulation of RDP reductase synthesis, deletion analysis of the smallest regulated nrd restriction fragment was performed. pCKT8 was digested with SaiI, and treated with Bal3I for various times. BamHI linkers were then ligated to the linear plasmid DNA, the products were circularized with T4 ligase and transformed into strain Ni 10. Transformants were screened both for the ability to induce galK activity upon thymine starvation and for the size of the BamHI - EcoRI fragment present. All transformants obtained in this procedure were able to regulate galK activity the same as pCKT8, even though up to 79 bp had been deleted (data not shown). pCKT300, the shortest clone, was then digested with BamHI, treated with exonuclease HI and S1 nuclease for various times, digested with EcoRI and the small fragments ligated into pKOTWl at the SnaI and EcoRI sites. Transformants were screened and analyzed as for the Bal31 procedure to produce the data presented in Figure 5. The exact endpoints of the clones assayed were identified by DNA sequence analysis (Sanger et al., 1977, Figure 6). Results presented in Figure 5 indicate that the fusions fall into three phenotypic regulatory classes. Class 1, (pCKT8, 229, 300) the original phenotype in which galK activity is low but inducible upon thymine starvation to 10 times the initial value; class 2, (pCKT308, 324-326) in which galK activity at all times during induction, as well as in uninduced cells, is 5 times that of class 1; and class 3, (pCKT25, 302, 307, 327) in which galK activity is low and cannot be induced by thymine starvation (Figures 5 and 6). Deletion of nrd DNA past nucleotide -124 relative to nrdP (pCKT307, class 3) clearly removes the regulatory response to thymine starvation, while deletion up to nucleotide -139 (pCKT325, class 2) appears not to affect inducibility of the construct. It appears, therefore, that the upstream border of the site required for response to thymine 1079 -

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starvation (regulatory site 2) is bounded by nucleotides - 139 and -124 (Figure 6, sequences present in class 1 and 2 but not class 3 fusions). A clone with approximate intermediate phenotype is pCKT310, whose deletion endpoint (-129) maps inside this region. During thymine starvation, the increase in rate of galK expression in this clone is somewhat lower than that seen for longer fragments, but significantly higher than the uninducible clone pCKT307. Inspection of the sequence in this area shows several intriguing regions (Figure 6). There are two 1 l-bp nearly perfect direct repeats in the sequence 5' to nrdP at -128 to

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- 117 and -67 to -57, one of which partially maps inside site 2. Between these two repeats is a large inverted repeat, whose stem loop structure has a calculated (Tinoco et al., 1973) potential energy of -18.4 kcal (Figure 6). Deletion of sequences upstream of site 2 (see Figures Sb and 6, clones pCKT308, 324-326) results in an 5-fold increased level in galK expression without affecting the ability to respond to thymine starvation. The upstream border of this region (regulatory site 1) maps between - 157 and -150 (Figure 6, sequences present in class 1 but not class 2 fusions). Deletion of

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