Differential mRNA stability controls relative gene expression within a polycistronic operon

Cell, Vol. 51, 1131-I 143, December

24, 1987, Copyright

0 1987 by Cell Press

Differential mRNA Stability Controls Relative Gene Expression within a Polycistronic Operon Sarah F. Newbury, Noel H. Smith,’ and Christopher F. Higgins Molecular Genetics Laboratory Department of Biochemistry University of Dundee Dundee DDl 4HN Scotland

Summary In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon. The polycistronic ma/EFG operon of E. coli contains two REP sequences (highly conserved inverted repeats) within the ma/E-ma/F intercistronic region. Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream ma/f mRNA, but also results in a g-fold reduction in the synthesis of MalE protein. A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA. The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common. Introduction In prokaryotic cells, genes that encode related functions are frequently organized as polycistronic operons. Such organization simplifies the coordinate induction and repression of these genes but makes differential expression of individual genes within an operon more difficult to achieve. As each gene in a polycistronic operon is transcribed from the same promoter, differential expression must be accomplished by transcriptional termination within the operon or, more commonly, by differences in the efficiency with which the individual genes are translated into protein. Differential translation of a single mRNA species could be achieved by differences in the relative strengths of the ribosome binding sites (e.g., the atp operon; McCarthy et al., 1985). Alternatively, differential expression might be effected by differential rates of mRNA degradation. In the last 3 or 4 years it has become increasingly apparent that differences in mRNA stability play an important role in determining the level of gene expression (reviewed by Higgins and Smith, 1986; Brawerman, 1987). Different mRNAs have very different rates of decay, and in many cases, the half-lives of specific mRNA species vary with environmental changes in a manner that is consistent with a role for mRNA stability in the control of gene expression (von Gabain et al., 1983; Nilsson et al., 1984; Belasco et * Present address: Department Rochester, New York 14627.

of Biology, University

of Rochester,

al., 1985; Collins et al., 1986; Bechhofer and Dubnau, 1987). However, the factors that determine RNA stability and their precise role in regulating gene expression are still largely obscure. In E. coli, two enzymes are primarily responsible for the bulk degradation of mRNA, RNAase II and polynucleotide phosphorylase (Kaplan and Apirion, 1974; Deutscher, 1985; Donovan and Kushner, 1986). These enzymes are 3’-5’exonucleases, and indeed, no 5’-3’ exoribonuclease activity has yet been identified in E. coli. Thus, sequences that impede the processive action of these 3’-!Yexonucleases can specifically stabilize upstream mRNA. This is frequently accomplished by stable secondary (stem-loop) structures in the mRNA (Schmeisser et al., 1984; Hayashi and Hayashi, 1985; Mott et al., 1985; Mackie, 1987; Newbury et al., 1987). Other factors may also influence mRNA decay. The presence or absence of ribosomes and the rate of translation can alter mRNA stability (Gupta and Schlessinger, 1976; Schneider et al., 1978), probably as a consequence of ribosomes protecting mRNA from nucleolytic attack. Specific cleavage by endonucleases can expose new 3’ ends that serve as substrates for processive 3’-5’ exonucleases. The best example of this is the cleavage of the major leftward transcript of phage h by RNAase III, which renders the upstream int mRNA more susceptible to exonuclease degradation (Guameros et al., 1982; Schmeisser et al., 1984). Endonuclease cleavage in intercistronic regions of polycistronic operons like lac and gal may also be important in the initiation of decay (Blundell and Kennell, 1974; Lim and Kennell, 1979; Cannistraro et al., 1986). Specific sequences located at the 5’end of an mRNA molecule can play a role in determining its stability (Gorski et al., 1985; Belasco et al., 1986; Bechhofer and Dubnau, 1987; Mackie, 1987). It has been suggested that the mechanism by which this is achieved may involve endonucleolytic cleavage (Belasco et al., 1986), possibly altering mRNA decay indirectly by affecting translation. Alternatively, 5’sequences may serve as binding sites for specific proteins that act as stability determinants. Might differential mRNA stability determine the relative expression of genes within a polycistronic operon? For a number of operons, differential stability of segments of mRNA encoding individual genes has been observed. Furthermore, in several cases there is a good correlation between the stability of different segments of mRNA and the relative amounts of the individual proteins produced from them in vivo (Burton et al., 1983; Blumer and Steege, 1984; Belasco et al., 1985; Bdga, 1987; Newbury et al., 1987). Such data have led to the suggestion that differential stability of segments of mRNA may regulate relative gene expression within an operon (Belasco et al., 1985). However, in none of these cases has such a mechanism been demonstrated directly. Interpretation of such data are complicated by the fact that the functional half-life of an mRNA molecule (the length of time for which that molecule is proficient in directing protein synthesis) is not

Cell 1132

REP 2

REP 1

4 b Lruu m!u I,” I “I”, 1 ~rr,,s,cc..~r,,,~~,~,~..,,~~~~~,,~~~~~~,~,.~~~~,,~,~~~~~~,.~....~~~..~~~~.~~~.~~~~~~.~“~~.~~~=~~.~~~~~~~.~~.~~~.~~~~“~~~~~.~..~~~~,~...~~~~,.,~~~,~~,~~..,~~~~,.~.,~~~~.,~ mr .,OIl.ThrL”. .*. “.I A 1 C.CIECI..C.CCL.TT..T~~,~,~.,,,~~~~~.,~~~~~~~~..~~~~~~~~~~~~~~~.~....~~~...~~~.~~~~ T7.ICCCTSSTTC..,T.~T..T..SCSS.TT 63

C.CICLI~.C.SS..~T..~~~,~~~..,,~~~~~.T~CCCCC,C

Figure 1. Sequences

of Deletions in the ma/E-ma/F

Cl,.rCCCICCTTC,.TT.(~..~.“~~~~,,~ lntergenic

Region

The top line is the sequence of the wild-type ma/E-ma/Fintergenic region of E. coli including the last 5 codons of ma/E and the first codon of ma/F (taken from Froshauer and Beckwith, 1984, and confirmed during our sequencing of this region). The two REP sequences are indicated by arrows above the sequence. The locations of the Stul and Hinfl sites used in constructing and sequencing the deletions are indicated. The two lower lines show the sequences of Al and A3, the open boxes indicating the extents of the two deletions. The sequences were determined by the procedure of Maxam and Gilbert (1980) with fragments end-labeled at the Hinfl site.

necessarily the same as its chemical half-life; functional inactivation and the degradation of mRNA can be temporally distinct processes (Schwartz et al., 1970; Gupta and Schlessinger, 1976; Chanda et al., 1985). Furthermore, it has been established for several operons that a proportion of stabilized mRNA is often translationally inactive (Hayashi and Hayashi, 1985; Mackie, 1986; Newbury et al., 1987). The mechanisms of translational inactivation are not well understood, although they may involve endonucleolytic cleavage at or near the ribosome binding site (Shen et al., 1981; Cannistraro and Kennell, 1985; Cannistraro et al., 1988; Portier et al., 1987). Nevertheless, the fact that translational inactivation can occur in vivo means that factors that stabilize mRNA and cause mRNA accumulation do not necessarily lead to an increase in protein synthesis. One important determinant of mRNA stability in E. coli and Salmonella typhimurium is the REP sequence, which is present in 500 to 1000 copies on the E. coli chromosome (Newbury et al., 1987). REP sequences are highly conserved inverted repeats that have the potential to form stable stem-loop structures in mRNA (Higgins et al., 1982; Gilson et al., 1984; Stern et al., 1984). Furthermore, REP sequences are generally transcribed into mRNA, either in the 3Qmtranslated sequences at the end of a transcription unit or in the intercistronic regions of multicistronic operons. In all cases that have been studied, REP sequences increase the half-life of upstream mRNA, apparently impeding the action of processive 3’-5’exonucleases, while having little or no effect on downstream mRNA (Newbury et al., 1987). Thus, when located in an intercistronic region of a polycistronic operon, REP sequences specifically stabilize upstream mRNA causing it to accumulate. We have previously found that, in the malEFG operon of E. coli, upstream ma/E mRNA is considerably more stable than is full-length ma/EFG mRNA (Newbury et al., 1987). The ma/EFG operon is one of two divergent operons that comprise the ma/6 locus encoding the maltoselmaltodextrin transport system (Figure 9: see Hengge and Boos, 1983, for a review of this transport system). The ma/E gene encodes an abundant periplasmic protein, present in a 20-to 40-fold excess over the membrane-associated MalF and MalG proteins (Koman et al., 1979; Shuman et al., et al., 1985). Thus, in this operon there 1980; Manson

is a good correlation between the relative stabilities of ma/E and ma/F mRNA and the relative abundance of their respective protein products. In addition, two copies of the REP sequence, in inverted orientation with respect to each other, are located in the ma/E-ma/F intergenic region (Froshauer and Beckwith, 1984). This led us to suggest that the REP sequences might be responsible for the increased stability of upstream ma/E mRNA and that this might account, at least in part, for the differential expression of genes within this operon (Newbury et al., 1987). In this paper we demonstrate that this is indeed the case. We show that the REP sequences are entirely responsible for the stability of upstream ma/E mRNA and that the differences in stability between ma/E and ma/F mRNA contribute substantially to differential gene expression within this operon. These data provide a direct demonstration that differential mRNA stability can play an important role in determining the relative expression of genes within a polycistronic operon. Results Construction of Deletions in the ma/E-ma/F lntergenic Region The maltoselmaltodextrin transport system of E. coli is encoded by five genes organized as two divergently transcribed operons (see Experimental Procedures). The malE-malF intergenic region of the malEFG operon contains two copies of the REP sequence in inverted orientation with respect to each other (Figure 1). To study the effects of the REP sequences on relative gene expression within this operon, we constructed deletions of the ma/E-ma/F intergenic region using Ba131 nuclease (illustrated and described in Experimental Procedures). Two deletions that were confined to the malE-malF intergenic region and did not extend into the adjacent structural genes were characterized in detail and sequenced (Figure 1). One of these deletions removed both REP sequences from the ma/f-ma/F intergenic region (83; plasmid pWJ173), while the other deletion (Al; plasmid pWJ171) removed just one of the two REP sequences. These two plasmids were used for subsequent experiments and were maintained in strain CH1596 (red AmalE444) to prevent recombination with the wild-type chromosomal operon. The construction of this strain is described in Experimental Procedures.

Differential 1133

mRNA Stability and ma/fFG

(A)

Expression

5

1

(8)

s +AA Xl

(Cl

y2

x2

Yl

recombination between Y, and Y2

recomblnahon between XT and X2

v,s ID)

8 ‘o+ t5i

8 5 ;! -65

kb

ab I, Figure 2. Integration of amalE-ma/F mosome

12.3 kb 2 0 kb lntergenic Deletion info the Chro-

(A) A recombinant h phage containing the 5.8 kb Bglll fragment from the ma/EFG operon (see Figure 9A) was lysogenized by homologous recombination with the chromosomal ma/&G operon of MC4100. The solid black bars indicate the chromosomal ma/ operon, the hatched bars indicate the ma/ sequences cloned into the h vector, and the thin line indicates the phage L genome. A solid circular flag indicates the wild-type ma/E-ma/F intergenic region, while a triangular flag indicates the intergenic region with both REP sequences deleted (A3). The abbreviations for restriction sites are: Bg, Bglll; S, Stul. (6) Diagram showing the product of the recombination event indicated in (A), assuming recombination occurs 3’ (to the right) of the ma/Ema/F intergenic region. Because the cloned 5.8 kb Bglll fragment includes 800 bp of DNA to the 5’ (left) side of the ma/E-ma/f intergenic region and 5000 bp to the 3’ side, recombination will occur about 6 times more frequently 3’ to the intergenic region; the lysogen used for additional experiments was shown to have this configuration by Southern blot analysis (data not shown). The product of this recombination is a merodiploid with two adjacent copies of the ma/EFG region, one wild-type and the other containing the ma/E-ma/F intergenic deletion. (C) The I lysogen is forced to deintegrate, which occurs by homologous recombination between the two copies of the malUG region. This recombination can occur either 5’ (to the left) of the REP sequences (between regions X1 and X,) or 3’ (to the right) (between Y, and Y,). Recombination between Y, and Yp, which occurs at about 6 times the frequency as recombination between X, and Xp, restores the wild-type chromosomal sequences. Recombination between X, and Xz results in replacement of the wild-type ma/E-ma/f intergenic region with the 96 bp deletion. The product of such a recombination (CH1602) is identical in all respects to MC4100 except that the 96 bp deletion of the ma/E-ma/F intergenic region from pWJ173 (A3; Figure 1) has been incorporated into the chromosomal operon. (D) Southern blot showing correct integration of the ma/E-ma/F deletion into the chromosome. Chromosomal DNA from MC4100 and one

Integration of a ma/E-ma/F Deletion onto the Chromosome Because of possible artifacts that might result from studies of the malEFG operon in multicopy (and, indeed, do occur; see below), the 96 bp deletion of the ma/E-ma/F intergenic region that removes both REP sequences (A3) was recombined into the chromosomal operon. This was achieved by cloning the plasmid-borne ma/EFG operon containing the deletion (A3) into a phage h derivative and forcing integration of the recombinant h phage into the chromosome (illustrated in Figure 2 and described in detail in Experimental Procedures). Since the recombinant h phage contains no att site, lysogens most frequently arise by homologous recombination with the chromosomal ma/EFG operon. A single such lysogen (CH1601) was induced by growth at 42% forcing the h phage to excise. Excision occurs by homologous recombination between the duplicate copies of the ma/B region in one of two ways, either restoring the wild-type ma/E-ma/F intergenic region or replacing wild-type chromosomal sequences with the 96 bp ma/E-ma/F deletion (see Figure 2). Derivatives in which the deletion was precisely incorporated into the chromosome were identified by Southern blot analysis (Figure 2D). One such derivative, CH1602, in which the 96 bp ma/E-ma/F intergenic deletion (A3) had replaced the normal chromosomal sequences was used for all subsequent studies. Sequences in the ma/E-ma/F lntergenic Region Are Responsible for the Accumulation of Upstream ma/E mRNA To examine the effect of deleting the malE-malf intergenic region on the accumulation of ma/E mRNA, strain CH1602 (A3) and its parent, MC4100, were grown in M63-maltose medium and RNA was isolated. Figure 3A shows a Northern blot of this RNA, probed with a 1223 bp Hinfl fragment spanning the ma/E gene. In the parental strain (MC4100) an abundant 1300 nucleotide mFtNA species extending from the operon promoter to the ma/E-ma/F intergenic region was detected. This mRNA species, which potentially encodes the MalE protein, is entirely lacking from strain CH1602, which is isogenic with MC4100 except for the 96 bp malE-malF intergenic deletion. We have demonstrated previously that this 1300 nucleotide mRNA species accumulates because it is more stable than full-length ma/EFG mRNA (Newbury et al., 1967). The data above demonstrate that this stability and the consequent ac-

of the h-sensitive derivatives after integration and excision (CH1602) was digested with Stul, separated by electrophoresis, and probed using the 5.8 kb Bglll fragment from the pEJ1. The parental strain MC4100 shows three Stul fragments: the 2.2 kb fragment arises as a result of cleavage at the Stul site in the ma/E-ma/F intergenic region; the 4.2 kb and 6.5 kb fragments are sequences to the left and right of the REP sequences, respectively. CH1602 is a result of recombination between X1 and Xp: the 2.2 kb and 4.2 kb fragments are missing and are replaced by a 6.4 kb band, showing that a ma/E-ma/F deletion of the correct size has been introduced onto the chromosome and that phage 1 has been lost. Recombination between Y, and Y2 would give a wild-type pattern.

Cdl 1134

0

(51

2

4

6

81012

(a) MC4100 (2 REPS)

(b) CH1602 (OREPS)

-

(c) pEJ1 (2 REPS) I

(d) pWJ171 (1 REP) .. (e) pWJ173 (0 REPS) Figure 4. Translational

malEFG -

malE -I)

Figure 3. Northern Operon of E. coli

mat E -

Slots of mRNA

Transcribed

from the ma/EFG

(A) Chromosomally encoded mRNA. RNA was isolated from MC4100 and its derivative CH1602. These two strains are isogenic except for a 96 bp deletion that removes the REP sequences from the ma/E-ma/F intergenic region of the CH1602 chromosome. A 1300 nucleotide, REPstabilized ma/E transcript from MC4100 is indicated; this transcript is absent from CH1602, although the loading of mRNA in the two tracks was identical. We have described the characterization of transcripts from the ma/EFG operon previously (Newbury et al., 1967). The exposure of this particular autoradiogram is insufficient to see the fulllength ma/EFG mRNA, which is much less abundant than the ma/Especific transcript (6; Newbury et al., 1967). (6) Plasmid-encoded mRNA. RNA was isolated from strain CH1596 (AmalE444) with no plasmid and from the same strain harboring pEJ1 (the wild-type ma/E-ma/F intergenic region containing both REP sequences), pWJl7l (pEJ1 with one of the REP sequences deleted), or pWJ173 (pEJ1 with both REP sequences deleted). The Northern blot was probed using a 1223 bp Hinfl fragment from pEJ1 that spans the ma/E gene (see Figure 9A). The full-length ma/EFG (3600 nucleotide) and REP-stabilized ma/E (1300 nucleotide) transcripts are indicated. The REP-stabilized ma/E transcript derived from pWJl7l is slightly shorter than that derived from the parental plasmid pEJ1 because of the 59 bp deletion introduced into pWJl7l. The faint ma/E-length band visible in the pWJ173 track is due to hybridization to intermediates in degradation, which artifactually accumulate at this point on the gel due to overloading with rRNA (see Newbury et al., 1967).

cumulation of this upstream ma/E-specific mRNA species are due to the REP sequences located in the malEFG intergenic region. We also examined the effect of deleting the ma/E-ma/F intergenic REP sequences from the plasmid-encoded malEFG operon. mRNA from strain CH1596 (Alma/E444 recA) and from the same strain containing plasmid pEJ1 (wild-

Inactivation

-

-'. of ma/E mRNA

Cells were grown in M63 amino acids medium with 0.4% maltose to an ODwo of 0.35, and rifampicin (200 uglml) was added. Aliquots (0.5 ml) were taken at time 0 and at the indicated time intervals (min) after the addition of rifampicin and were pulse-labeled with [%]methionine. The MalE protein was immunoprecipitated. Samples were separated by electrophoresis, and the labeled MalE protein was detected by autoradiography. This procedure gives a measure of the decrease in rate of MalE synthesis after arrest of transcription and, hence, a measure of the rate of translational inactivation of ma/E mRNA. (a and b) MalE synthesis from the chromosomal ma/EFG operon of strain MC4100 and from its derivative, CH1602, which is deleted for the REP sequences in the ma/E-ma/F intergenic region. (c, d, and e) MalE synthesis from strain CH1596 (AmaE444) harboring pEJ1 (wild-type), pWJl7l (one REP deleted), or pWJ173 (both REPS deleted), respectively. It should be noted that the amount of MalE protein made in a 45 set pulse at time 0 is less for CH1602 than for MC4100. This is because the deletion in CH1602 results in a lower rate of MalE synthesis at steady-state growth (see text). Densitometry was carried out on various exposures of these and similar autoradiographs to obtain accurate figures for the half-life of translational inactivation.

type ma/EFG operon), pWJl7l (one REP deleted), or pWJ173 (both REPS deleted) was examined by Northern blot analysis (Figure 38). As expected, the host strain, CH1596, which is deleted for the chromosomal ma/E gene, gave no hybridization signal. When plasmid pEJ1 (encoding the wild-type ma/EFG operon) was introduced into CH1596, a 1300 nucleotide RNA species accumulated, similar to that encoded by the chromosomal operon. Small amounts of full-length ma/EFG mRNA could also be detected. When both REP sequences in the ma/E-ma/F intergenic region were deleted (pWJ173) the 1300 nucleotide mRNA species could no longer be detected. Thus, the 96 bp malE-malf deletion has a similar effect on mRNA stabilization whether present on a multicopy plasmid or integrated into the chromosome. Interestingly, however, when just one of the two REP sequences was deleted from the ma/E-ma/F intergenie region (Al) accumulation of the 1300 nucleotide ma/E mRNA species was unaffected. A single copy of the REP sequence is apparently as effective as two copies in stabilizing upstream mRNA. REP Stabilized ma/E mRNA Can Direct Protein Synthesis The 1300 nucleotide transcript stabilized by the REP sequences can potentially encode the MalE protein. If stabilization of this transcript plays a role in regulating

Differential 1135

mRNA Stability and ma/EFG Expression

h

I

&Z LamB-MalE --

MC4100

--

CH1602 E 1

bottom

-----+

top

Figure 5. Effect of Deleting REP Sequences on the Steady-State Rate of MalE Synthesis Cells of strains MC4100 (wild-type ma/EFG operon) and CH1602 (deleted for the chromosomal ma/E-F intergenic region) were grown in M63-maltose, pulse-labeled, and immunoprecipitated simultaneously with anti-MalE and anti-Lam6 antibodies. The immunoprecipitated proteins were separated by electrophoresis and detected by autoradiography. The MalE and Lam6 proteins are indicated. Lam6 expression is unaffected by the ma/E-ma/f deletion and serves as an internal control. The difference in the rate of MalE synthesis in the two strains was quantitated by densitometry of this and other similar autoradiographs, normalized using the LamB standard. Densitometric scans of the MalE bands that have been normalized with respect to amount of LamB are shown next to the gel.

differential expression within the ma/EFG operon, it is important to demonstrate that the stabilized mRNA is translationally active and can direct MalE synthesis. Having deleted the REP sequences from the ma/E-ma/F intergenie region, this could be tested directly. Growing cells were treated with rifampicin to inhibit RNA synthesis. At specified time intervals after rifampicin treatment, aliquots of cells were pulse-labeled for 45 set with [35S]methionine, MalE protein was immunoprecipitated from extracts of the labeled cells, and the immunoprecipitate was separated by SDS-PAGE. This procedure gives a measure of the decrease in rate of MalE synthesis after mRNA synthesis is inhibited (i.e., the rate of translational inactivation of ma/E mRNA) (Figure 4). Figure 4a shows that synthesis of MalE protein from the wild-type operon in MC4100 continues for at least 12 min after rifampicin treatment; densitometry shows that half-life for translational inactivation of ma/E mRNA to be about 7 min. In contrast, the half-life for translational inactivation of ma/E mRNA from strain CH1602, in which the ma/E-ma/F REP sequences are deleted, is only about 2 min (Figure 4b). Thus, deletion of the REP sequences not only destabilizes ma/E mRNA, but also decreases the half-life for translational inactivation of ma/E mRNA. This demonstrates unambiguously that at

least a proportion of the MalE protein synthesized in vivo must be translated from REP-stabilized mRNA. Deletion of REP Sequences Decreases the Rate of MalE Synthesis The fact that the REP sequences in the malE-malF intergenie region stabilize upstream ma/E mRNA in a translationally active form implies that the REP sequences play a role in determining the level of MalE protein in the cell. If this is the case, then the steady-state rate of MalE synthesis from the wild-type operon would be expected to be greater than that from the operon with the REP sequences deleted. To accurately assess the contribution of the REPstabilized ma/E mRNA during normal growth, cells in exponential growth were pulse-labeled with [35S]methionine and cell lysates were immunoprecipitated simultaneously with anti-LamB and anti-MalE antibodies. The chromosomally encoded LamB protein serves as a control for variations in 3% uptake and sample loading. Precipitated proteins were separated by SDS-PAGE, and the amount of MalE protein was determined by densitometry of the resulting autoradiograms. Data were obtained for four independent samples for each strain and were normalized by comparison with the Lam6 internal control. Figure 5 shows that the rate of MalE synthesis (i.e., the amount of protein made in a 45 set pulse) from MC4100 is 5 to 6-fold greater than that made from CH1602 (in which the two REP sequences are deleted). Thus, the REP sequences in the ma/E-ma/F intergenic region clearly play a role in determining the rate of MalE synthesis. It is possible that the steady-state rate of MalE synthesis is influenced by feedback inhibition of MalE translation. If this were the case, the 5 to 6-fold difference in the rate of MalE synthesis between the wild-type strain (MC4100) and the deletion strain CH1602 may actually underestimate the effect of REP-dependent mRNA stabilization on MalE synthesis. We therefore examined the rate of MalE synthesis immediately after induction of the maltose operon, before ma/E mRNA and MalE protein have accumulated to their steady-state levels. Figure 6 shows the rate of MalE synthesis at time 0 and at 2 min intervals after adding maltose and CAMP to the growing cells. These data show that at each time point the rate of MalE synthesis (i.e., the amount of protein made in a 45 set pulse) is 5 to 6-fold greater for MC4100 than for CH1602. In addition, the increase in rate of MalE synthesis (i.e., the slope of the lines in Figure 6) is also about 5- to g-fold greater for MC4100 than for CH1602. Thus, the effects of the REP sequence on MalE synthesis are essentially the same, regardless of the amount of MalE protein or ma/E mRNA in the cell. Differential Expression within the Maltose Operon Is Regulated by the REP Sequences The data presented above demonstrate that deletion of the chromosomal REP sequences reduces the rate of MalE synthesis 5- to 6-fold. To determine whether this difference in rate is reflected in the total amount of MalE protein accumulated by the cell, proteins from cells in exponential growth were separated by SDS-PAGE and the

Cell 1136

+ *

MC4100 CH1602

minutes after induction. Figure 6. MalE Synthesis

during Induction

of the ma/EFG Operon

Ceils of MC4100 (wild-type ma/Ef-G operon) and CH1602 (96 bp chromosomal ma/E-ma/F deletion) were grown in M63 amino acids medium with glucose as a carbon source. At time 0, expression of the maltose operon was induced by adding maltose (0.4%) and CAMP (5 mM) to the growing cells. Samples were taken at the indicated times after induction, pulse-labeled with [35S]methionine, and immunoprecipitated with anti-MalE antibody. The labeled MalE protein was separated by electrophoresis and detected by autoradiography. The amount of protein synthesized in a 45 set pulse at each time point was quantitated by densitometric scanning. These data were plotted. They give a measure of the increasing rate of MalE synthesis immediately following induction. A value of 100% translation is defined as the rate of MalE synthesis from MC4100 during steady-state growth in maltose, and all other values are expressed relative to this figure.

relative amounts of MalE protein were estimated by densitometry. Figure 7A shows that the total amount of MalE protein in CH1602 (deleted for the REP sequences) is g-fold less than for the parental strain MC4100. This O-fold

(A)

difference in MalE accumulation is slightly but reproducibly greater than the 5 to 6-fold difference in the rate of MalE synthesis for the two strains; the reason for this is not known. A similar g-fold reduction in MalE protein was found for periplasmic fractions isolated from CH1602 and Mc4100 (data not shown). Thus, it is clear that the REP sequences play an important role in regulating differential expression within the ma/EFG operon. The effect of deleting the REP sequences on MalE accumulation is also reflected in the less intense colony color of CH1602 on MacConkey-maltose plates when compared with its otherwise isogenic parent, MC4100. This indicates that the g-fold reduction in MalE protein is sufficient to’reduce maltose utilization. This is consistent with a previous demonstration that, when MalE is reduced to less than 20% of its wild-type level (while the other components remain at their wild-type levels), it becomes limiting for maltose transport (Manson et al., 1965). Furthermore, even a small reduction in the level of MalE reduces the efficiency of chemotaxis toward maltose (Manson et al., 1965). Thus, the high level of MalE protein maintained by the REP sequences is physiologically important. Effect of Deleting the REP Sequences from the Plasmid-Encoded ma/EFG Operon The experiments described above show that the REP sequences in the ma/EfG operon play an important role in maintaining a high level of MalE synthesis. In these experiments we were careful to examine regulation of the operon in its normal chromosomal context. However, when we examined the role of the REP sequences in the malEG operon cloned onto a multicopy plasmid, significant differences were observed. We showed above that deletion of the 96 bp ma/E-ma/F intergenic region, remov-

Figure 7. Effect of Deleting the REP Sequences on the Total Cellular Level of MalE Protein

(B)

(A) Chromosomally encoded proteins. Coomassie-strained gel showing total cell proteins isolated from strains MC4100 (wild-type ma/EFG operon) and CH1602 (deleted for the REP sequences within the chromosomal ma/E-ma/F intergenic region). (6) Plasmid-encoded proteins. Coomassiestained gel showing total cell proteins from CH1596 (AmaE444) and from CH1596 harboring pEJ1 (wild-type malEFG operon), pWJ171 (one REP deleted), or pWJ173 (both REP sequences deleted). The plasmid-encoded MalE and p-lactamase proteins are indicated. Duplicate extracts from each strain are shown.

Mal E

P

Lac tamase

Differential 1137

mRNA Stability and ma/EFG Expression

pEJ1

=,z a Lam BMal E-m

rr*

Ix h 1

pWJ171

pWJ173

bottom B Figure 6. Effect of ma/E-ma/F Multicopy Plasmid

Deletions

on MalE Synthesis

top from a

Strain CH1596 (AmalE444) harboring pEJ1 (wild-type ma/EFG operon), pWJ171 (one REP deleted), or pWJ173 (both REPS deleted) was grown in M63 maltose medium to mid-log phase. Samples (0.5 ml) were pulse-labeled, immunoprecipitated with both anti-MalE and anti-Lam6 antibodies, and separated by electrophoresis. The labeled proteins were detected by autoradiography. The MalE and LamB proteins are indicated. Faint bands above MalE and Lam6 are unprocessed forms of the two proteins. LamB is chromosomally encoded and serves as an internal control. The rate of translation of LamB from CH1596 is lower than from MC4100 and CH1602 (Figure 5) because of the TnlO insertion in the chromosomal me/K-/amS intergenic region of CH1596, which decreases Lam6 expression by about 75% (Brass and Manson, 1964). Densitometric scans of the MalE bands, normalized with respect to amount of chromosomally encoded LamB, are shown.

ing both REP sequences, eliminates accumulation of the stable 1300 nucleotide ma/E transcript in either the chromosomal or the plasmid-encoded operon. The effect of this deletion on the translational inactivation of ma/E mRNA was also similar whether plasmidencoded or chromosomally encoded (compare Figures 4c and 4e with 4a and 4b). However, when the effect of the deletion on the rate of MalE synthesis was measured, a clear difference was seen between plasmid and chromosome. The rate of MalE synthesis at steady state (i.e., the amount of MalE protein made during a 45 set pulse at steady-state growth) was only about e-fold lower for pWJ173 (REP sequences deleted) than for the parental plasmid, pEJ1 (Figure 8).

This difference was not due to plasmid copy number (data not shown) and contrasts with the 5- to 6-fold reduction in the rate of MalE synthesis caused by the identical deletion on the chromosome (Figure 5). This finding was also reflected in the total amount of MalE protein accumulated by strains carrying pEJ1 and pWJ173 (Figure 78). Unlike the chromosomal operon, where deletion of the REP sequences caused a g-fold reduction in the amount of MalE in the cell, the same 96 bp deletion causes only a 3-fold reduction in MalE protein when plasmid-encoded (compare pEJ1 with pWJ173). Thus, the effect of the ma/E-ma/F intergenic deletion on upstream MalE synthesis is artificially low when examined in a multicopy genetic context. A Single REP Sequence Is as Effective as Two in Stabilizing Upstream mRNA and Increasing Upstream Protein Synthesis The ma/EFG intergenic region contains two REP sequences. The experiments described above were carried out in strains in which both of the REP sequences had been deleted. However, in many operons REP sequences occur in single copy. To determine whether a single REP sequence is able to stabilize translationally active mRNA, we examined the effects of a deletion of just one of the REP sequences (Al ; pWJ171). Figure 38 shows that a single copy of the REP sequence is apparently equally effective in stabilizing upstream mRNA as are two copies; deletion of one of the REP sequences (pWJ171) does not significantly reduce accumulation of the 1300 nucleotide mRNA species. Consistent with this result, the deletion of just one of the two REP sequences has no detectable effect on the rate of translational inactivation of ma/E mRNA (Figure 4), on the rate of MalE synthesis (Figure 8), or on the amount of MalE protein accumulated by the cell (Figure 78). Thus, a single REP sequence is apparently as efficient as two REP sequences in stabilizing upstream mRNA and increasing upstream gene expression. Discussion The protein products of polycistronic operons are often synthesized in very different amounts, yet the factors determining their relative expression are not well understood. In some operons the efficiencies of ribosome binding and translational initiation are clearly important. In this paper we present direct evidence for an alternative mechanism for effecting differential gene expression: the stability of specific segments of mRNA. The ma/EFG operon of E. coli encodes three proteins required for the transport of maltose across the cytoplasmic membrane. The first of these proteins, MalE, is highly expressed compared with the two promoter-distal proteins in the operon, MalF and MalG. We showed previously that, in this operon, a specific segment of mRNA encoding MalE (but not MalF or MalG) has a longer half-life than fulllength ma/EFG mRNA and hence is specifically accumulated (Newbury et al., 1987). The accumulation of this ma/E-specific transcript, together with the excess of MalE protein in the cell compared with MalF and MalG, sug-

Cell 1138

gested that differential gene expression within this operon might be due to this specific stabilization of ma/f mRNA. Furthermore, two copies of the REP sequence are located in the ma/E-ma/F intergenic region. Since we have shown for other operons that REP sequences can stabilize upstream mRNA (Newbury et al., 1987) it seemed possible that REP-dependent stabilization of upstream mRNA may be responsible for differential gene expression within this operon. In this paper we have tested this hypothesis directly. Deletion of the REP sequences from the ma/Ema/F intergenic region on the chromosome was found to reduce the stability of upstream mRNA such that ma/Especific transcripts no longer accumulate. This demonstrates that the intergenic REP sequences are indeed responsible for increasing the stability of upstream mRNA. This REP-stabilized mRNA was shown to be translationally active. Furthermore, deletion of the REP sequences from the ma/E-ma/F intergenic region not only eliminated accumulation of ma/E mRNA but also reduced the total accumulation of MalE protein in the cell by about g-fold. As these experiments were carried out in the normal chromosomal context, there can be no doubt that, at least in this operon, differential mRNA stability plays a major role in determining relative gene expression. How general is such a regulatory mechanism? REP sequences are present in 500 to 1000 copies on the E. coli and S. typhimurium chromosomes and are frequently found in intergenic regions between two genes that are differentially expressed (Higgins et al., 1982; Gilson et al., 1984; Stern et al., 1984). Although the REP sequence is apparently the only stable stem-loop structure that occurs in multiple copies on the E. coli chromosome (Maurice Hofnung, personal communication), stem-loop structures of different sequence are frequently found between genes that are differentially expressed (e.g., Belasco et al., 1985; Hiles et al., 1987). Because many stable stem-loop structures in mRNA serve as barriers to 3-5 exonucleases (Guptaet al., 1977; Mott et al., 1985; Hayashi and Hayashi, 1985; Hiles et al., 1987; Newbury et al., 1987) it seems reasonable to assume that many, if not all of these intergenie stem-loop structures increase the stability of upstream mRNA. Thus, differential mRNA stability mediated by intergenic stem-loop structures could potentially provide a rather widespread mechanism for the differential regulation of gene expression. Such a model was first suggested to account for differential expression within the rxcA operon of Rhodopseudomonas (Belasco et al., 1985). Belasco et al. (1985) showed that a 5’segment of the rxcA transcript, encoding two light harvesting polypeptides, is considerably more stable than the 3’ segment encoding the reaction center subunits. The stability of the two segments of transcript correlated well with the relative amounts of their respective protein products. Furthermore, potentially stable stem-loop structures were identified in the intergenic region, which could well be responsible for the differential stability of transcripts. Similar correlations between mRNA stability and the relative levels of the respective protein products have been reported for other operons (e.g., Burton et al., 1983; Blumer and Steege, 1984; Br%ga, 1987). However,

increased mRNA stability does not necessarily lead to increased expression of upstream genes. First, it appears that not all potentially stable stem-loop structures serve to stabilize upstream mRNA (Wong and Chang, 1986). Second, even in operons in which upstream mRNA is stabilized, the stable mRNA is not necessarily translationally active. For example, in the histidine transport operon of S. typhimurium, the REP sequences between hi.sJ and hisQ stabilize upstream hisJ mRNA, yet they are certainly not responsible for the 3O:l ratio of HisJ to HisQ expression; deletion of the REP sequences from this intergenic region in the chromosome abolishes the accumulation of hisJ-specific mRNA, yet reduces HisJ synthesis by at most 2-fold (Stern et al., 1984; Newbury et al., 1987). In this operon some other factor, probably ribosome binding, must be responsible for differential expression (Newbury et al., 1987; G. F.- L. Ames, personal communication). Thus, although a role for mRNA stability in regulating relative gene expression within operons is probably quite widespread, it will be difficult to predict whether it is generally important until the factors that influence stability and the translational inactivation of mRNA are better understood. The high level of MalE protein due to the downstream REP sequences is of physiological importance to the cell, and in strains deleted for the REP sequences, the level of MalE protein becomes limiting. The MalE protein is a periplasmic maltose-binding protein that functions as primary receptor for both transport and chemotaxis. A high level of the periplasmic component is important for the function of binding protein-dependent transport systems, and in all systems so far examined, the periplasmic component is present in large excess over the membraneassociated components (Ames, 1986; Hiles et al., 1987). In addition, it has been shown that even a small reduction in the amount of MalE protein, while the other components are maintained at a constant level, can be limiting for both transport and chemotaxis (Manson et al., 1985). Thus, the REP-dependent stabilization of ma/E mRNA ensures that the MalE protein is present in sufficient quantities for efficient operation of the maltose transport and chemotactic systems. Although REP-dependent stabilization of ma/E mRNA can account in large part for the difference between upstream and downstream gene expression in the malEFG operon, other factors must also be involved. The ratio of the MalE to MalF proteins has been estimated to be 20to 40-fold, while differential mRNA stability accounts for only a O-fold difference in their expression. The remaining difference is presumably accounted for by a difference in ribosome binding and translational efficiency between ma/E and ma/F mRNA. The superimposition of two independent mechanisms for achieving different levels of expression may circumvent potential difficulties in achieving large differences by a single mechanism. For example, it may be difficult to achieve a 30-fold difference in expression simply by differences in ribosome binding efficiency. We suggested previously that the effects of the REP sequences on the ratio of upstream to downstream gene expression within an operon can vary depending on the rate

Differential 1139

mRNA Stability and ma/EFG Expression

of transcription (Newbury et al., 1987). At low rates of transcription, the ratio of products will also be low. Thus, an additional difference in ribosome binding efficiency will ensure that, for an inducible operon such as ma/EFG, the MalE protein is always made in excess over MalF and MalG. While it is quite clear that the REP sequences play an important role in regulation of the ma/EFG operon in its normal chromosomal context, somewhat surprisingly their effect is much reduced when the operon is encoded on a multicopy plasmid. Thus, deletion of the REP sequences from chromosomal ma/EFG operon reduced the level of MalE protein by about g-fold, whereas the identical deletion in the plasmid-encoded operon only reduced MalE expression by 2- to 3-fold. This difference does not seem to be due to differences in stability between plasmid-encoded and chromosomally encoded mRNA and is therefore presumably due to differences in translational efficiency. It is not difficult to imagine that, as considerably more ma/E mRNA is transcribed from the multicopy plasmid than from the chromosome, factors other than the level of ma/f mRNA may become limiting for MalE synthesis. The increased concentration of malE mRNA in plasmid-containing cells may also result in a higher proportion of this mRNA being translationally inactivated. Whatever the mechanism, this finding illustrates the importance of studying regulation in the normal chromosomal context and the potential artifacts that can arise when studies are solely confined to genes cloned on multicopy plasmids. REP sequences appear to stabilize upstream mRNA wherever they are located by impeding the activity of 3’5’ exonucleases (Newbury et al., 1987). Indeed, we have demonstrated that, even in vitro, REP sequences can block the processive action of 3’-5’ exonucleases, precluding a requirement for a specific protein to mediate their protective function (R. McLaren, S. F N., and C. F. H., unpublished data). An internal stem-loop structure forming the rho-independent terminator trpt has also been shown to impede the progress of RNAase II in vitro (Mott et al., 1985). The ma/E-ma/F intergenic region contains two REP sequences in inverted orientation with respect to each other (Froshauer and Beckwith, 1984). In other locations on the chromosome, REP sequences occur either singly or in up to four adjacent copies (Gilson et al., 1984; Stern et al., 1984). It is not yet clear what the significance, if any, of multiple copies might be. Wherever two adjacent copies of the REP sequence are found, they are always in inverted orientation with respect to each other. This provides potential for the formation of alternative stem-loop structures. As each REP sequence is itself an inverted repeat, the two REP sequences could fold independently to form two distinct stem-loop structures separated by an unpaired region of 15 nucleotides. Alternatively, one REP sequence could base pair intramolecularly with the other to form a single stable structure (Higgins et al., 1982). It is also conceivable that intermolecular base pairing occurs between REP sequences on different mRNA molecules, as has been found for other regulatory systems (Mizuno et al., 1984; Simons and Kleckner, 1983). We do

not know which of these alternative foldings predominates in vivo. Significantly, we have found here, by deletion of just one of the REP sequences from the ma/E-ma/F intergenie region, that the single remaining REP sequence is as efficient as the original two REP sequences at enhancing mRNA stability and gene expression. Similarly, it has been found for the rxcA operon that stem-loop structures with very different AGs of formation can stabilize upstream mRNA to very similar extents (Belasco et al., 1985). At first sight it would seem reasonable to assume that the larger and more stable stem-loop structure (involving both REP sequences; AG = -60.4 kcallmol) would delay an RNAase for longer than the less stable single REP structure (AG = -23.5 kcallmol). The finding that a single REP sequence is equally effective in stabilizing upstream mRNA as are two REP sequences has implications for the mechanism by which the 3’-5’exonucleases overcome the barrier provided by stem-loop structures; the overall stability of a stem-loop structure is not the only factor important in determining its efficiency at blocking 3’-5’ exonuclease activity. This will be discussed in detail elsewhere. If a single REP sequence is as efficient as two REP sequences at stabilizing upstream mRNA and increasing upstream gene expression, why do REP sequences often occur in groups of two or more adjacent copies? It is, of course, possible that under certain growth conditions two REP sequences are more efficient than one at stabilizing upstream mRNA. More probably, their ability to stabilize upstream mRNA may not be the primary factor leading to the maintenance of these sequences on the chromosome. REP sequences may serve an alternative function that provides the primary selection for their maintenance, such as chromosomal organization (Higgins et al., 1982; Stern et al., 1984; Gilson et al., 1986). Alternatively, REP sequences may be conserved by gene conversion events rather than by providing a positive selective advantage to the cell (Newbury et al., 1987). This does not preclude the possibility that some copies of the REP sequence, such as those in the malE-malF intergenic region, have been recruited by the cell to serve specific functions, but does explain a number of observations about the properties, location, and distribution of the REP sequences (Newbury et al., 1987). The role of gene conversion in maintenance of the REP sequences is currently being tested.

Experimental Procedures Bacterial Growth Unless indicated, all of the E. coli strains used were derivatives of MC4100 (araD139 rpsLi50 deoC7 A(argWac)U769 retA ptsF25 rpsR flbs307; Casadaban, 1976). Strain JM63 (ara A(lac-pmAt3) rpsL Q60dlacZ AM15 Vieira and Messing, 1962) was used as a host for the pUC-based plasmids, facilitating the screening for inserts using X-gal indicator (5-bromo-4-chloro-3-indolyl+D-galactoside). Strain LE392 (hsdR514 (rmk, m+k) supE44 supF58 lacy1 galK2 galT22 met81 trpR55 tonA; Murray et al., 1977) was used for manipulations involving phage h. Cells were grown with aeration at 37W, except for h lysogens, which were grown at 30%. LB (Roth, 1970) and M63 (Miller, 1972) were used as complete and minimal media, respectively. Phage I. lysates were prepared in TB medium as described by Silhavy et al. (1964).

Cell 1140

IA) pEJ1 + pWJ160 + lam B

msl K

mal E

mal F

mal G

1 kb

Figure 9. Construction Region

of Deletions

in the m&E-malF

lntergenic

(A) Map of the malB locus of E. coli. The malEFG and malK-lam8 operons, which encode the components of the maltose/maltodextrin transport system, are transcribed divergently from a central regulatory region and have been sequenced in their entirety (Clement and Hofnung, 1981; Gilson et al., 1982; Duplay et al., 1984; Froshauer and Beckwith, 1984; Dassa and Hofnung, 1985). The promoters and direction of transcription are shown by arrows. The REP sequences in the ma&mslf intergenic region are indicated by a solid circular flag. The DNA fragments from the maltose operon cloned in plasmids pEJ1 and pWJl80 are also shown. The Sal1 site at the 3’ end of ma/K was converted to a BamHl site during construction of pEJ1 (E. Davis and P. Henderson, unpublished data). The DNA probes used for Southern and Northern blot analyses are indicated and are discussed more fully in the text. 8, BamHI; Bg, Bglll; H, Hinfl; E, EcoRI; Sa, Sall; Sm, Smal; s, stu1. (B) Strategy for construction of Ba131 deletions of the REP sequences from the ma/f-ma/F intergenic region. Details are given in the text and in Experimental Procedures. Vector sequences are indicated by the thin line; ma/B sequences, by the thick line. The extent of the ma/B region cloned in pEJ1 and pWJl80 is shown in Figure I. The REP sequences in the ma/E-ma/F intergenic region are indicated by a solid circular flag. Where a deletion of the REP sequences has been introduced is indicated by a triangular flag. bla and cat indicate the P-lactamase (Amp’) and chloramphenicol acetyltransferase (Cmlr) genes, respectively. Abbreviations for restriction endonuclease cleavage sites are as given above. Plasmids are not drawn to scale.

DNA and RNA Techniques Restriction endonucleases, Ba131, and DNA-modifying enzymes were purchased from Amersham, Bethesda Research Laboratories, or New England Biolabs and were used according to the manufacturers instructions. Plasmid DNA was prepared by the alkaline lysis procedure (Birnboim and Daly, 1979) and, when necessary, was purified by cesium chloride density gradient centrifugation. E. coli chromosomal DNA and phage h DNA were prepared as described by Silhavy et al. (1984). Labeling DNA with [32P]dCTP by nick translation, agarose gel electrophoresis, and Southern transfers and hybridization were as described by Mania& et al. (1982). RNA extraction using hot phenol, electrophoresis in formaldehyde gels, and Northern blot analysis have been described previously (Newbury et al., 1987). Determination of Plasmid Copy Number Relative plasmid copy number was determined by one of two alternative methods. For overnight cultures, copy number was determined as described by Nugent et al. (1986). Plasmid DNA was isolated by the miniprep method of Kado and Liu (1981) and separated on a 1% agarose gel. The relative copy number was quantified by densitometry. For cultures at log phase, plasmid DNA was isolated using the alkaline procedure (Birnboim and Daly, 1979). Plasmid DNA was linearized with BamHl and separated on a 1% agarose gel. The amount of DNA was quantified by densitometric scanning. For each determination at least four independent replicates were analyzed.

Construction of a AmalE recA Strain To study the expression of ma/E from various plasmids, we constructed a rsc4 derivative of a strain deleted for the chromosome1 ma/E gene to preclude recombination between the plasmid and chromosome. MM317 is a derivative of MC4100 that carries a large nonpolar deletion in malE (AmalE444; Shuman, 1982). In addition, this strain has a Tn70 transposon inserted in the ma/K-/am6 intergenic region (zjb729::TnIO), which allows MaIT-independent expression of LamB at about 25% of the normal ma/F level (Brass and Manson, 1984). To make a recA derivative of MM317, a h phage containing a mutant recA gene (1N1183; kindly provided by Noreen Murray) was used to replace the wild-type reed gene. This phage also contains the clgs7 temperature-sensitive repressor mutation. Strain MM317 was lysogenized with IN1 183 at 30°C. Since this k phage lacks the affsite, lysogens can only arise by homologous recombination between the recA genes of the phage and chromosome. Putative tysogens were plated in the presence of )rclh80A(a&inf)9, which contains the same 1 repressor as XN1183 but uses TonA rather than LamB as a receptor, to screen against h’ mutations (Silhavy et al., 1984). Lysogens were grown at 42OC to induce deintegration of the lysogenic I. phage. This occurs by homologous recombination between the two copies of rscA gene in the lysogen, such that the I phage and one copy of the mcA gene are eliminatad leaving either the wild-type or the mutant recA gene. To identify the derivatives that had lost the 5 and retained the mutant rec4 gene, the colonies surviving at 42OC were screened for sensitivity to phage

Differential 1141

mRNA Stability and malUG

Expression

A and for the presence of the recA mutation by their sensitivity to ultraviolet irradiation (Silhavy et al., 1984). One such strain, CH1596 (AmaE recA zjb-729:TnlO), was used for all subsequent studies. Construction of ma/E-ma/F Deletions The construction of deletions in the ma/f-ma/F intergenic region is illustrated in Figure 9. Plasmid pEJ1 contains an 6.3 kb DNA fragment, encoding the ma/EFG operon, cloned into pBR326 (Figure 9A). To construct deletions within the ma/E-ma/F intergenic region, we wished to make use of the Stul site located in this intergenic region. As pEJ1 contains two Stul sites, we constructed a plasmid in which only the ma/E-ma/F Stul site was present by cloning the 3 kb BamHI-Smal fragment from pEJ1 between the BamHl and Smal sites of pUC8 (see Figure 9B). The resulting plasmid, pWJl60 (Figure 9B), was digested to completion with Stul, and treated with Bal31 nuclease. Any ragged ends were filled in using the Klenow fragment of DNA polymerase, and the plasmids were recircularized by ligation. To screen derivative plasmids for deletions. DNA was initially digested with EcoRl and Bglll to identify those with a slight reduction in the size of the 1112 bp Bglll-EcoRI fragment. Plasmids with a small deletion were further screened for deletion size by digesting with Hinfl and EcoRI. Two deletions that appeared to be of the appropriate size to remove the REP sequences but not to extend into the m&or ma/Fstructural genes (Al and A3; Figure 1) were sequenced by the chemical degradation method (Maxam and Gilbert, 1980), labeling the DNA at the Hinfl site in ma/E (Figure 9B). To incorporate these deletions into an intact ma/EFG operon, the BamHI-Smal fragments of plasmids carrying the deletions (pWJ161 and pWJl63) were cloned back into pEJ1, replacing the wild-type BamHI-Smal fragment (Figure 98). Plasmids carrying these deletions (pWJ171 and pWJl73, respectively) were maintained in strain CH1596 (see above) to avoid recombination with the AmalEfecA the chromosomal malEFG operon. Integration of a ma6ma/F Deletion onto the Chromosome The plasmid-borne ma/E-ma/F deletion (A3; pWJ173) was integrated onto the E. coli chromosome, replacing the wild-type copy of the intergenie region as illustrated in Figure 2. The 5.8 kb Bglll fragment from pWJ173, containing the ma/EFG operon with the 96 bp ma/E-ma/F intercistronic deletion (A3), was cloned into hL47clss7 (Loenen and Brammer, 1980), replacing the BamHl stuffer fragment. Recombinant phage were identified by plaque hybridization (Maniatis et al., 1982) using the 2.2 kb Stul fragment from pEJ1 (Figure 9A) as probe. One such recombinant phage was used to lysogenize strain MC4100 as described above for IrecA. As the katf site is deleted from this phage, lysogens can only arise by homologous recombination between the cloned ma/ fragment and the chromosomal ma/ operon (Figure 2A). Lysogenization at the ma/B locus was confirmed by transducing the lysogen to tetracycline resistance using a Pi lysate of MM31 7 that carries a TnlO in ma/B: a high proportion of the resulting transductants became X-sensitive. One MC4100 lysogen (CH1601) was grown at 42%, forcing the 1 to deintegrate by homologous recombination, either leaving the wild-type ma/S region or replacing the wild-type chromosomal sequences with the ma/E-ma/F deletion (Figure 2C). To identify those derivatives that had acquired the deletion and to confirm loss of the h DNA, chromosomal DNA was extracted from the X-sensitive derivatives and screened by Southern blot analysis for loss of the 2.2 kb Stul fragment, using the 5.8 kb Bglll fragment as probe (Figure 2D). This 2.2 kb Stul fragment is characteristic of the wild-type ma/EFG operon but would be absent from strains carrying the A3 deletion in which the Stul site from the ma/E-ma/F intergenic region is missing. Strains in which the A3 deletion had integrated into the chromosome could also be distinguished from the wild-type by their colony color on MacConkey-maltose plates (see Results). One such derivative was used subsequently and named CH1602. Although the Southern blot shows only that the integration event is precise, it cannot detect any point mutation (i.e., in the ribosome binding site) that may have arisen during the manipulations. However, this is highly unlikely as integration occurred at the expected frequency, as there is no reason to suppose that point mutations should arise at anything more than the normal low frequency during these manipulations and because independent recombinants behaved in a fashion identical to CH1602. Furthermore, we have shown that the ribosome binding site is unaltered using diag-

nostic restriction shown).

enzymes

that cleave at these sequences

(data not

Cell Fractionation, Immunoprecipitation, and Gel Electrophoresis Total cell proteins were visualized by resuspending 0.16 ml of a mid-log culture of cells in 10 ~1 of loading buffer and separating by electrophoresis on a 10% SDS-polyacrylamide gel (Laemmli. 1970; Ames, 1974). Gels were stained with Coomassie blue. Periplasmic fractions were isolated as described previously (Higgins and Hardie, 1983). When required, cells were pulse-labeled with (%]methionine aqd labeled products were immunoprecipitated as described previously (Newbury et al., 1987). After electrophoresis, the gel was fixed and dried, and the labeled proteins were detected by autoradiography. Photographs of Coomassie-stained gels or autoradiographs were analyzed by densitometry using an LKB 2190-001 Gelscan with an Apple II 2202 Ultrascan interface and software. Acknowledgments We are grateful to Robert McLaren, Andy Flavell, James McClellan, and Noreen Murray for helpful discussions. We thank Bill Brammer, Hannes Brass, Peter Henderson, Mike Manson. and Noreen Murray for providing phage, plasmids, bacterial strains, or antibodies. This work was supported by grants from the Medical Research Council to C. F. H. C. F. H. is a Lister Institute Research Fellow. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received August 3, 1987; revised September

24, 1987.

References Ames, G. F.- L. (1974). Resolution of bacterial proteins by acrylamide gel electrophoresis on slabs. J. Biol. Chem. 249, 634-644. Ames, G. F.- L. (1986). Bacterial periplasmic transport systems: structure, mechanism and evolution. Ann. Rev. Biochem. 55, 397-425. Biga, M. (1987). Transcriptional organization; regulation and coordination of gene expression in the biogenesis of Escherichia co/i PAP pili. Umee University Medical Dissertations. New Series 193 (Umei, Sweden: University of Umei). Bechhofer, D. H., and Dubnau, D. (1987). Induced mRNAstabilityin cillus subtilis. Proc. Natl. Acad. Sci. USA 84, 498-502.

Ba-

Belasco, J. G., Beatty, J. 1, Adams, C. W., von Gabain, A., and Cohen, S. N. (1985). Differential expression of photosynthetic genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript. Cell 40, 171-181. Belasco, J. G., Nilsson, G., von Gabain, A., and Cohen, S. N. (1986). The stability of E. coli gene transcripts is dependent on determinants localized to specific mRNA segments. Cell 48, 245-251. Birnboim, H. C., and Daly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. Blumer, K. J., and Steege, D. A. (1984). mRNA processing in Escherichia co/i: an activity encoded by the host processes bacteriophage fl mRNA’s. Nucl. Acids Res. 72, 1847-1861. Blundell, M., and Kennell, D. (1974). Evidence for endonucleolflic attack in decay of lac messenger RNA in Escherichia co/i. J. Mol. Biol. 83, 143-161. Brass, J. M., and Manson, M. D. (1984). Reconstitution of maltose chemotaxis in Escherichia co/i by addition of maltose binding protein to calcium treated cells of maltose regulon mutants. J. Bacterial. 757, 881-890. Brawerman, 48, 5-6.

G. (1987). Determinants

of messenger

RNA stability, Cell

Burton, Z. F., Gross, C. A., Watanabe, K. K., and Burgess, R. R. (1983). The operon that encodes the sigma subunit of RNA polymerase also

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encodes ribosomal 32, 335449.

protein 521 and DNA primase in E. coli K12. Cell

Cannistraro, V. J., and Kennell, D. (1985). The 5’ ends of Escherichia co/i lac mRNA. J. Mol. Biol. 182, 241-248. Cannistraro. V. J., Subbarao, M. N., and Kennell, D. (1966). Specificendonucleolytic cleavage sites for decay of Escherichia co/i mRNA. J. Mol. Biol. 192, 257-274. Casadaban, M. J. (1976). Transposition and fusion of the lac genes to selected promoters in E. co/i using bacteriophage lambda and Mu. J. Mol. Biol. 104, 541-555. Chanda, P K., Ono, M., and Kung, H.- F. (1985). Cloning, sequence analysis and expression of alteration of mRNA stability gene (Ams) of f. co/i. J. Bacterial. 767, 446-449. Clement, J. M., and Hofnung, M. (1981). Gene sequence of the lam6 receptor, an outer membrane protein of E. coli K-12. Cell 27, 507-514. Collins, J. J., Roberts, G. P, and Brill, W. J. (1986). Posttranscriptional control of KlebsieNa pneumoniae nif mRNA stability by the nifL product. J. Bacterial. 168, 173-178. Dassa, E., and Hofnung, M. (1985). Sequence of gene ma/G in E. co/i K-12. Homologies between integral membrane components from binding protein-dependent transport systems. EMBO J. 4, 2287-2293. Deutscher, M. P (1985). E. coli RNAases: soup. Cell 40, 731-732.

making sense of alphabet

Donovan, W. F!, and Kushner, S. R. (1986). Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia co/i K-12. Proc. Natl. Acad. Sci. USA 83, 120-124. Duplay, P., Bedouelle, H., Fowler, A., Zarbin, I., Saurin, W., and Hofnung, M. (1984). Sequences of the ma/E gene and of its product, the maltose binding protein of Escberichia co/i K-12. J. Biol. Chem. 259, 10606-10613. Froshauer, S., and Beckwith, J. (1984). The nucleotide sequence of the gene for ma/F protein, an inner membrane component of the maltose transport system of Escherichia co/i. J. Biol. Chem. 259, 10896-10903. Gilson, E., Nikaido, H., and Hofnung, M. (1982). Sequence of the ma/K gene in E. co/i K-12. Nucl. Acids Res. 10, 7449-7458. Gilson, E., Clement, J.- M., Brutlag, D., and Hofnung, M. (1984). A familyof dispersed repetitive extragenic palindromic RNA sequences in E. coli. EMBO J. 3, 1417-1422. Gilson, E., Pernin, D.. Clement, J. M., Szmelcan, S., Dassa, E., and Hofnung, M. (1986). Palindromic units from E. coli as binding sites for a chromoid-associated proten. FEBS Lett. 206, 323-328. Gorski, K., Roth, J.- M., Prentki, P, and Krisch, H. M. (1985). The stability of bacteriophage T4 gene 32 mRNA: a 5’ leader that can stabilize mRNA transcripts. Cell 43, 461-469. Guameros, G., Montanez, C., Hernandez, T., and Court, D. (1982). Posttranscriptional control on bacteriophage h int gene expression from a site distal to the gene. Proc. Natl. Acad. Sci. USA 79, 238-242. Gupta, R. S., and Schlessinger, D. (1976). Coupling of rates of transcription, translation and messenger ribonucleic acid degradation in streptomycin-dependent mutants of Escherichia co/i. J. Bacterial. 725, 84-93. Gupta, R. S., Kasai, T., and Schlessinger, D. (1977). Purification and some novel properties of RNAase II. J. Biol. Chem. 252, 8945-8951. Hayashi, M., and Hayashi, M. (1985). Cloned DNA sequences that determine mRNA stability of bacteriophage @X174 in vivo are functional. Nucl. Acids Res. 13, 5937-5948. Hengge, R., and Boos, W. (1983). Maltose and lactose transport in Escherichia co/i. Examples of two different types of concentrative transport systems. Biochim. Biophys. Acta 737, 443-478. Higgins, C. F., and Hardie, M. M. (1983). Periplasmic protein associated with the oligopeptide permeases of Salmonella fyphiftwium and Escherichia co/i. J. Bacterial. 155, 1434-1438. Higgins, C. F., and Smith, N. H. (1986). Messenger RNA processing, degradation and the control of gene expression. In Regulation of Gene Expression, S.G.M. Symposium Vol. 39, I. R. Booth and C. F. Higgins, eds. (Cambridge: Cambridge University Press), pp. 179-198. Higgins, C. F., Ames, G. F- L., Barnes, W. M., Clement,

J. M., and

Hofnung, M. (1982). A novel intercistronic yotic operons. Nature 298, 766-762.

regulatory element of prokar-

Hiles, I. D., Gallagher, M. P., Jamieson, D. J., and Higgins, C. F. (1987). Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J. Mol. Biol. 795, 125-142. Kado, C. I., and Liu, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. J. Bacterial. 145, 1365-1373. Kaplan, Ft., and Apirion, D. (1974). The involvement of ribonuclease I, ribonuclease II and polynucleotide phosphorylase in the degradation of stable ribonucleic acid during carbon starvation in E. co/i. J. Biol. Chem. 249, 149-151. Koman, A., Harayama, S., and Hazelbauer, G. L. (1979). Relation of chemotactic response to the amount of receptor: evidence for different efficiencies of signal transduction. J. Bacterial. 738, 739-747. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lim, L. W., and Kennell, D. (1979). Models for decay of Escherichia co/i lac messenger RNA and evidence for inactivation cleavages between its messages. J. Mol. Biol. 735, 369-390. Loenen, W. A. M., and Brammer, W. J. (1960). A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 20, 249-259. Mackie, G. A. (1987). Posttranscriptional regulation of ribosomal tein S20 and stability of the S20 mRNA species. J. Bacterial. 2697-2701.

pro769,

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Manson, M. D., Boos, W., Bassford, P J., and Rasmussen, B. A. (1985). Dependence of maltose transport and chemotaxis on the amount of maltose binding protein. J. Biol. Chem. 260, 9727-9733. Maxam, A. M., and Gilbert, W. (1980). Sequencing end-labelled DNA with base specific chemical cleavages. Meth. Enzymol. 65, 499-560. McCarthy, J. E.G., Schairer, H. U., and Sebald, W. (1985). Translational initiation frequency of atp genes from Escherichia co/i: identification of an intercistronic sequence that enhances translation. EMBO J. 4, 519-526. Miller, J. H. (1972). Experiments in Molecular Genetics Harbor, New York: Cold Spring Harbor Laboratory).

(Cold Spring

Mizuno, T., Chou, M.. and Inouye, M. (1984). A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (mic RNA). Proc. Natl. Acad. Sci. USA 87, 1966-1970. Mott, J. E., Galloway, J. L., and Platt, T. (1985). Maturation of Escherichia co/i tryptophan operon mRNA: evidence for 3’ exonucleolytic processing after rho-independent termination. EMBO J. 4, 1887-1891. Murray, N. E., Brammer, W. J., and Murray, K. (1977). Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 750, 53-61. Newbury, S. F., Smith, N. H., Robinson, E. C., Hiles, I. D., and Higgins, C. F (1987). Stabilization of translationally active mRNA by prokaryotic REP sequences. Cell 48, 297-310. Nilsson, G., Belasco, J. G., Cohen, S. N., and von Gabain, A. (1984). Growth rate dependent regulation of mRNA stability in Escherichia co/i. Nature 372, 75-77. Nugent, N. E., Smith, T. J., and Tacon, W. C. A. (1986). Characterisation and incompatibility properties of ROM-derivatives of pBR322-based plasmids. J. Gen. Microbial. 132, 1021-1026. Portier, C., Dondon, L., Grunberg-Manago, M., and Regnier, P (1987). The first step in the functional inactivation of the Escherichia co/i poly nucleotide phosphorylase messenger is a ribonuclease Ill processing at the 5’ end. EMBO J. 6, 2165-2170. Roth, J. R. (1970). Genetic techniques lism. Meth. Enzymol. 77a, 3-35.

in studies of bacterial metabo-

Schmeisser, V., McKenney, K., Rosenberg, M., and Court, D. (1984). Removal of a terminator structure by RNA processing regulates int gene expression. J. Mol. Biol. 176, 39-53.

Differential 1143

mRNA Stability and ma/EFG Expression

Schneider, E., Blundell, M., and Kennell, D. (1976). Translation mRNA decay. Mol. Gen. Genet. 760, 121-129.

and

Schwartz, T., Craig, E., and Kennell. D. (1970). Inactivation and degradation of messenger ribonucleic acid from the lactose operon of Escherichia coli. J. Mol. Biol. 54, 299-311. Shen, V., Cymamon, M., Daugherty, B., Kung, H.-F., and Schlessinger, D. (1981). Functional inactivation of lac-peptides mRNA by a factor that purrfies with fscherichia co/i RNAase Ill. J. Biol. Chem. 256, X396-1902. Shuman, f-f. A. (1962). Active transport Chem. 257, 5455-5461.

in Escherichia

coli K-12. J. Biol.

Shuman, H. A., Silhavy, T. J., and Beckwith, J. R. (1960). Labellmg of proteins with 6-galactosidase by gene fusion. J. Biol. Chem. 255, 168-174. Silhavy, T. J., German, M. L., and Enquist, L. W. (1984). Experiments with Gene Fusions (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Simons, R. W., and Kleckner, N. (1983). Translational transposition. Cell 34, 663-691.

control of IS10

Stern, M. J., Ames, G. F.- L., Smith, N. H., Robinson, E. C.. and Higgins, C. F. (1964). Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell 37, 1015-1026. Vieira, J., and Messing, J. (1962). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259. von Gabain, A., Belasco, J. G., Schottel, J. L., Chang, A. C. Y., and Cohen, S. N. (1963). Decay of mRNA in Escherichia co/i: investigation of the fate of specific segments of transcripts. Proc. Natl. Acad. Sci. USA 80, 653-657. Wong, H. C., and Chang, S. (1966). Identification of a positive retroregulator that stabilises mRNAs in bacteria. Proc. Natl. Acad. Sci. USA 83, 3233-3237.