J. Mol. Biol. (1991) 219, 623-634

In Viva Interaction of Escherichia coli lac Repressor N-Terminal Fragments with the lac Operator Anastasia M. Khoury’* ‘Department

t, Harry S. Nick2 and Ponzy Lu’.$

of Chemistry, University of Pennsylvania Philadelphia, PA 19104, U.S.A.

‘Department of Biochemistry and Molecular Biology University of Florida, Gainesville, FL 32610, U.S.A. (Received

15 June 1990; accepted 28 February

1991)

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequenceencoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/ promoter. In addition to the wild-type lacl gene sequence, single amino acid substitutions were generated at positions 3 (Pro3 -+ Tyr) and 61 (Ser61 + Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10’ to 104. The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced /l-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain. Keywords: gene regulation; repressor: operon

1. Introduction For three decades, since Jacob t Monod (1961a,b) first used the Escherichia wli lactose metabolism system to investigate the problem of enzyme adaptation, the E. coli lactose operon has been the paradigm of gene regulation. This operon, with its regulatory elements, is the prototypical system for many genetic and biochemical studies (Miller & Reznikoff, 1980). Its repressor, a tetrameric protein of 360 amino acid residues per subunit (Farabaugh, 1978), yields two domains per subunit upon limited proteolytic cleavage. The N-terminal 60 or so residues display DNA binding properties while the

t Present address: Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138, U.S.A. $ Author to whom correspondence should be addressed.

C-terminal 300 residue portion remains tetrameric and displays inducer binding properties (Platt et al., 1973). Considerable genetic evidence (Miller & Reznikoff, 1980; Miller, 1984; Kleina & Miller, 1990) and in vitro biochemical data (Ogata & Gilbert, 1978, 1979) indicate that these N-terminal amino acids comprise most if not all of the DNA interaction site. ‘H and 19F nuclear magnetic resonance experiments with N-terminal fragments show that operator DNA and non-specific DNA affect tyrosine ring positions differently (Nick et al., 1982; Arndt et al., 1982). This has been further supported by simultaneous compensatory changes in the lac operator DNA sequence and the amino acid residues of the N terminus of the Zac repressor (Ebright, 1985, 1986; Lehming et al., 1988, 1990). The exclusive DNA binding role of lac repressor N-terminal domain has been contradicted by in vitro data which suggests that the inducer-sensitive operator DNA binding specificity resides entirely in tetramers missing the first 51 or 59 amino acid residues (Matt,hews, 1979;

623 0022~2836/91/120623-12

$03.00/O

0

1991

~Acsdemia

Press

Limited

A. M. Khoury

O’Gorman et al., 1980; Dunaway & Matthews, 1980; Manly et al., 1983, 1984; Manly & Matthews, 1984). Because of its small size, the lac N-terminal fragment or “headpiece” was among the first proteins to be subjected to detailed ‘H nuclear magnetic resonance structural studies. A folded secondary structure for the first 51 amino acid residues has been proposed (Kaptein et al., 1985; Zuiderweg et al., 1983) that is similar to other helix-turn-helix DNA binding proteins (Anderson et al., 1982; Pabo & Sauer, 1984; Sauer et al., 1982; Takeda et al., 1983). It would be useful to expand on the solution structure by extending the length of the N-terminal sequence because the lac repressor is a classical allosteric protein. Correctly folded peptides with amino acid residues beyond 51 are of interest in the understanding of intersubunit communication as it, relates to DNA binding. In all previous work, the N-terminal DNA binding domain was isolated by cleavage with proteases such as clostripain, chymotrypsin and trypsin to yield N-terminal fragments of 51, 56 and 59 amino acid residues (Geisler & Weber, 1977). Proteolytic cleavage is limited by the specificity of available proteases and the fortuitous exposure of sensitive peptide bonds. Longer peptide fragments, those greater than 59 residues, with DNA binding activity could not be obtained by this method. Furthermore, the proteolytic method requires a number of isolation steps after digestion of the intact purified lac repressor resulting in low overall yield. As a consequence of the experiments described here, we can now obtain biologically active N-terminal lac repressor fragments, of any length, in yields adequate for physical chemical experiments. The interaction of sequence-specific proteins with target DNA sequencesis dependent on the composition of the ionic environment, including organic species such as glutamate (Leirmo et al., 1987; Record et aE., 1977; Richey et aE., 1987). Thus, the ultimate test of new N-terminal fragment lengths and variations of the lac repressor with the operator DNA sequence would be to use the in vivo footprinting method (Church & Gilbert, 1984; Nick &, Gilbert, 1985). In order to set up this test, the DNA sequence encoding various lengths of the lac repressor N-terminal domain were cloned into a vector system transcribed from the coliphage T7 RNA polymerase promoter (Tabor & Richardson, 1985). Because of the relatively high concentrations of lac repressor fragments accumulated, we were also able to detect a reduction in /3-galactosidase synthesis in the cell culture (Miller, 1972). An in vivo evaluation of the DNA binding characteristics of various altered lac headpiece peptides is also described here. In addition to generating the wild-type N-terminal fragments of 56 and 64 amino acid residues, single amino acid substitutions at positions 3 (Pro3 -+ Tyr) and 61 (Ser 61 --) Leu) as well as the double substitution in the 64 amino acid fragment have been introduced. These amino acid substitutions have been referred to as 112, X86 and

et al.

112-X86 (Schmitz et al., 1978). These substitutions were chosen becausethey increase the DNA binding affinity of the intact lac repressor by a factor of lo* for the single change and lo4 for the double change (Schmitz et al., 1978). Production of these lac repressor fragments have also been confirmed in vivo by radioimmunoassays. DNA binding charaeteristics of these protein fragments and their effect, on transcription have been verified by in vitro footprinting and whole-culture fl-galactosidase assay.

2. Materials

and Methods

(a) Plasmids and bacterial Plasmid

pT7-7 encoding

the T7

strains

Ixornoter

and plasmid

pGPl-2 encodingthe T7 RNA polymerase structural

gene

were provided by Tabor & Richardson (1985). Plasmid pT7-7 confers fl-lactamase resistance (Amp’) and contains the T7 RNA polymerase promoter, ~$10, upstream from the translational start site for the T7 gene 10 protein followed by a polylinker site (Fig. l(a)). Plasmid pGPl-2, approximately 7200 bpt in size, confers kanamycin resistance and contains the T7 RNA polymerase gene behind the A P, promoter, under the control of cT857 (a temperature sensitive 1 cI repressor). The 1 c1 gene, also encoded in pGPl-2, is transcribed from an E. coli Pl,, promoter. Our source of ZacZ gene fragments, pPL1, a derivative of pMT ZacZ (Brown et al., 1987), was a gift from Mylos Brown. It contains the Zucl gene behind the 3, t’,, promoter. E. coli DH9, an i- derivative of HBIOI. and HBlOl (F-Zaci+o’y-pro IPW, rpsL r;m, rec.4) were provided by Betz & Sadler (1978). (b)

DNA

manipulations

and clminy

procedures

DNA preparations, enzyme reactions and bacterial transformations were performed as described by Maniatis et al. (1982). Restriction endonucleases, DNA ligase and phage T4 polynucleotide kinase were obtained from Bethesda Research Laboratories and New England Biolabs. Mung bean nuclease was purchased from Pharmacia. (c)

lacl yene fragment

cloninq

Plasmid pPI,I was digested with P.stI and f&XI to remove 930 bp plus overhanging ends (Khoury. 1989) from the resident ZacZ gene. The resulting plasmid was religated using a 32 bp oligonucleotide linker that encoded amino acid residues 52 to 56 as well as 2 in-phase TAA stop codons. The resulting construct was digested with BarnHI and then treated with mung bean nuclease to remove protruding 5’ end. Subsequent digestion with PstI released a fragment 185 bp long encoding the 56 amino acid N-terminal domain of the Zac repressor gene. This fragment was inserted into the PstI and blunt-ended NdeI sites of pT7-7, a T7 RNA polymerase mediated vector (Fig. l(a)). The result’ant plasmid expression pTHP56wt was transformed into E. coli DH9 containing helper plasmid pGPl-2 encoding T7 RNA polymerase (Fig. l(b)). Plasmid pTHP56wt was used t,o produce pTHP64wt,> pTHP64leu61 and pTHP56tyr3 by cassette mutagenesis, while pTHP64tyr3 and pTHP64tyr3leu61 cassette mutagenesis from were produced via pTHP56tyr3 (Table 1). 7 Abbreviations used: bp, base-pair(s), isopropyl-I-thio-/?-n-galactoside.

IPTG.

625

E. coli RepressorInteraction with lac Operator (d) Growth and induction

(a)

Media used for maximal protein production contained 50 mM-potassium phosphate (pH 7.2), 2% (w/v) tryptone, 1% (w/v) yeast extract, 05% (w/v) NaCl, 62% (w/v) glycerol, 50 pg ampicillin and kanamycin/ml. Cells containing the 2 plasmids were grown in this enriched media with antibiotic selection at 30°C to A,,, = 1.2 to 1.4. At that time the temperature of the bacterial cultures was raised to 42°C where it was maintained for 25 min. The heat inactivates the ~1857 protein and allows the expression of T7 RNA polymerase. The temperature of the culture was shifted to 37°C and maintained at that temperature for 1 to 1.5 h to accumulate protein product produced by T7 RNA polymerase before harvesting the cells. For maximal protein production, rifampicin could be added to the culture when at 37°C (at a concentration of 100 pg/ml) to suppress bacterial gene expression by E. coli RNA polymerase. Rifampicin was not used in the assays here. For the footprinting, we wanted to avoid interference of an inactivated E. coli RNA polymerase on the DNA at the overlapping E. coli-Zac promoter-operator sequence. For the fi-galactosidase assay, E. coli RNA polymerase was needed to transcribe the /I-galactosidase gene.

lac N-terminal

O&OX

7.2 kb \

I’ ,’

PlSA origin

Kan r

J

,

\

\

, “\ ‘,

of bacteria

\\ , \ L

(e) Confirmation of lac repressor N-terminal peptide synthesis

Figure 1. T7 RNA polymerase promoter system expressing the Zac repressor N-terminal DNA binding fragments. All cells contained pGPl-2 plasmid (b), which is 7200 bp in length and expresses T7 RNA polymerase (Tabor & Richardson, 1985). Cells also contained one of the pTHP plasmids with ZacZ coding sequence fragment inserted into T7-7 (Tabor & Richardson, 1985) which express the appropriate length and/or altered Zae repressor fragment (a) (see Table 1). Start site for the Zac repressor gene is ATG instead of wild-type GTG for increased efficiency; rbs, ribosome binding site. (c) Schematic of the region on pGPl-2 examined in footprinting experiments. A 32P-labeled probe corresponding to the coding strand of the region of interest (between the HinfI sites) was made using a 31 base primer as shown extended on singlestranded M13mp18 with sequence (c) to amplify the signal. This allows monitoring of the methylation patterns on the DNA strand illustrated. kb, lo3 bases or base-pairs.

Zac repressor peptide production was confirmed by radioimmunoassay (Hunter, 1973, 1974). Serial dilutions of crude lysate were incubated with [1251]Bolton Hunter labeled Zac headpiece derived by chymotrypsin cleavage, polyclonal rabbit antibody against Zac repressor and sheep anti-rabbit antibodies. After incubation at 4°C the precipitate formed was spun out and the radioactivity in the pellet was compared with the input level by liquid scintillation counting. Cts/min were compared with a standard curve and the concentration of protein produced by cells with each of the plasmids are shown in Table 1. (fJ Amim

Protein fragment produced

pTHP56wt pTHP56tyr3 pTHP64wt pTHP64tyr3 pTHP641eu61 pTHP64tyr3leuGl

N-P3 N-Y3 N-P3

C C S614

sequence analysis

The DNA sequence of each of these constructs was confirmed by dideoxy DNA sequencing (Sanger et al., 1977) either by inserting the region to be sequenced into the Ml3 vector and using E. coli DNA polymerase (Klenow fragment) and the standard 15 base Ml3 primer following the protocol of Davis et al. (1986) or by doublestranded plasmid sequencing utilizing Sequenase (Haltiner et al., 1985; Hattori & Sakaki, 1986; Kraft et al., 1988). In addition, the amino acid composition of the 56 amino acid fragment from pTHP56wt was confirmed by acid hydrolysis and subsequent amino acid separation and

Table 1 Wild-type and mutant lac repressor

Plasmid

acid and DNA

fragments produced Relative

pg N-terminal produced/g wet cells

N-terminal fragment in

980+40 s86+70

80 70

23+14 56+16

110+10 450*60

10 30 30 20

39+13 14+2 23f12 20&l

N-Y3

S61X

N-P3

L61X

410*30

N-Y3

L61-C

299+20

CdS

(PM)

/3-galactosidase activity (%)

626

A. M. Khoury

quantitation. The sequence of the N-terminal 5 amino acids of this fragment was also confirmed by automated Edman degradation.

(g) In vivo footprinting The method of Church & Gilbert (1984) was used as described for the Zuc system (Nick & Gilbert, 1985). The Zac promoter-operator region (ZucU V5 promoter) on plasmid pGPl-2, which supplied the T7 polymerase, was used as the test DNA sequence for these experiments. This avoids complications of effects due to cyclic AMP binding protein and the need to examine induction in glycerol-based growth media. The concentration of the plasmid in the cell was 10-s nM, assuming a plasmid copy number of 15 to 20 (Sambrook et al., 1989). DH9 cells containing appropriate plasmids were grown as described above. Before harvesting the cells, they were cooled down to 4°C and treated with dimethyl sulfate (1.25 pi/ml) for 15 s. The reduced temperature was chosen because Ogata & Gilbert (1979) have shown that the effects are more pronounced in vitro at 0°C compared to room temperature. The reaction was stopped by pouring the cells into a IO-fold excess of ice-cold PBS (1 mm-KH,PO,, 15 mn+Na,HPO,, 2.7 mM-KCl, 137 mM-NaCl, pH 7.5) containing 70 mm-Tris. HCl (pH 7.4) after which the cells were spun down. Plasmid DNA was isolated, restrictiondigested with Hinff, piperidine-treated, denatured and run on a denaturing polyacrylamide gel. The DNA was then electrotransferred from the gel onto nylon membrane (DuPont GeneScreen) crosslinked by ultraviolet light and 32P-labeled sequence at high probed with the appropriate stringency (65°C and @5 M-sodium phosphate). The probe used was an Ml3 minus strand containing the N-terminal end of the ~1857 through the Zac promoter-operator and into the C terminus of the T7 polymerase sequence gene described below (see Fig. l(c)). Note that the Zac promoter on plasmid pGPl-2 is the ZacUV5 promoter in which positions - 8 and -9 are both A. T base-pairs instead of T. A and G.C, respectively (Gilbert, 1976; Hopkins. 1974). These changes make this promoter CAPindependent. Positive controls were performed in HBlOl (wild-type strain from which DH9 was derived) both with and without pGPl-2. In the case where pGPl-2 was not used, the footprint of the genomic operator-promoter was examined.

(h) Ml3

hybridization

probe

The E’coRI-P&I fragment from pGPl-2 containing the lac promoter-operator was subcloned into the EcoRI-P&I site of the M13mp18 vector. The P&I site in pGPl-2 is on the C-terminal side of the ~1857 gene, single-stranded phage DNA was isolated from this construct which was then used to make 32P-labeled Ml3 probes for hybridizations to visualize the footprint. The probe was made by primer extension using E. coli DNA polymerase, dGTP. dCTP, dTTP, [cr-32P]dATP and a 31 base primer with the following sequence: 5’.GGCGACGTGCGTCCTCAAGCTGCTCTTGTGT-3’. This primer abuts the 3’ end of the HinfI site closest to the ~1857 gene (Fig. l(c)). It begins 25 bases away from the start site of ~1857 and extends 31 bases into the gene (encodes amino acids 8 to 19: Hendrix et al., 1983). The extended strand along the single-stranded Ml3 template was separated from the parent strand by running the product on a denaturing gel and eluting the desired

et al. strand. This extension was necessary to make the probe radioactive enough to detect.

(i) nensitometry A Shimadzu densitometer was used for all densitometric scans. Medium sensitivity was used with single wavelength set at 560 nm. Peak areas were measured by weighing the paper under the peaks in triplicate. Lanes were normalized for fluctuations in DNA loaded on the gels by comparing peaks upstream from the Zac promoter region. For quantitative evaluation, the intensities of the adjacent guanine doublets, G + 11, G + 12, and G - 1, G - 2, were added together and treated as one peak each due to the lack of resolution. (j) p-Galactosidme

assay

HBlOl cells were grown as described above, except that when the temperature was shifted to 37”C, IPTG was added to a final concentration of 10m3 M. At the end of t,he 1.5 h period at 37”C, the cells were cooled to 4°C and diluted to AsOO = @2 to @6. /?-Galactosidase assays were performed with orthonitrophenol as described by Miller (1972). The control assay and the assays from the cells containing plasmids expressing t.he Zac repressor fragments contain 10 n&l-wild-type levels of tet.rameric Zac repressor that are held in the induced conformation with 10V3 M-exogenous IPTG.

3. Results (a) lac repressor

N-terminal

fragments

are produced

The synthesis and accumulation of the cloned protein fragment in the cell were confirmed by competition of our protein product, with ‘251-labeled proteolytically cleaved headpiece for lac repressor polyclonal antibodies in a radioimmunoassay. This assay was also used to optimize growth and timing of the induction procedure (inactivation of ~1857 followed by synthesis of T7 RNA polymerase to transcribe the lac repressor DNA sequence) for maximal Zac repressor N-terminal fragment yield. Table 1 shows typical yields of protein for the two lengths of headpiece and their corresponding altered sequences. The yields from the various clones ranged from 10 to 80 PM in viva. The concentration of peptide fragments in the cell were estimated assuming a volume for E. coli of I.7 x lOWI5 liters and a wet weight of 9.5 x lo-l3 g/cell (Neidhardt et al.. 1987). An additional assumption was t,hat> these Zac N-terminal peptides have similar affinities for the anti-lac* repressor antibodies. Both the wild-type, (HP56wt). and altered HP56tyr3 (Pro3 -+ Tyr) 56 amino acid genes seemed to yield more peptide per cell (about, 1 mg protein/g cells) than the 64 amino acid N-terminal fragment’ DNA. Cells containing plasmid pT7-7 without lacl sequences were used as a negative control. Tt is possible that the additional eight amino acid residues render the protein less stable and more susceptible to proteases. It has been found that some sequences at the C terminus of a protein

E. coli RepressorInteraction

can alter the stability of a protein (Bowie & Sauer, 1989). In addition, thermal stability of a protein is highly sequence-dependent, so that even a single substitution could drastically alter stability (Parse11 & Sauer, 1989). Fluctuations in yield observed here could be caused by increased susceptibility of some of these N-terminal protein fragments to proteases due to the nature of their primary or secondary structure, a process not yet fully understood. On SDS/polyacrylamide gels, the fragment lengths appear t’o be complete, as expected from the DNA sequence.

with

lac Operator

627

(a)

(b)

(b) In vivo protection of operator DNA sequence against chemical methylation DNA binding properties of the Zac repressor protein fragments were examined in vivo by dimethyl sulfate footprinting to compare the binding mechanism of the headpiece protein with the intact repressor. This procedure also tests the proper folding of the partial peptide sequence. The footprinting experiments compare the extent of methylof guanine N-7 by dimethyl sulfate ation permeating the cell wall in the presence and absence of the lac repressor peptide. If a protein is bound to a specific stretch of the DNA major groove, the guanine N-7 positions in that region are protected from methylation. The differential methylation observed reflects specific protein-DNA interactions. Non-specific binding is not detected since it appears as uniform DNA cleavage along the entire length of a DNA molecule when displayed on a sequencing electrophoretic gel (Ogata & Gilbert, 1978). The sequence we examined by the footprint procedure is the 1acUV5 promoter-operator region that is on plasmid pGPl-2 (Tabor & Richardson, 1985), which is also the source of T7 RNA polymerase. We chose to probe the plasmid since we anticipated a dissociation constant in the PM range for the lac repressor peptide interaction with fragment-operator as estimated by Ogata & Gilbert (1979) at 0°C. The region probed by DNA hybridization is that between two HinfI sites as shown in Figure l(c). A 31 base primer was used to make a Ml3 minus strand probe corresponding to about 140 bp right of the operator DNA to greater than 250 bp on the left. These extra sequences allow the accumulation of enough radioactivity to develop autoradiograms. Representative densitometer scans of the autoradiograms are shown in Figure 2 and a summary of t’he effects of all the wild-type and mutant headpieces from Table 1 are shown as bar graphs in Figures 3. 4 and 5. It was found that the lac repressor peptide fragments protect DNA specifically with a pattern similar to intact repressor. The results from this study are similar to those observed by Ogata & Gilbert (1978) in vitro for both intact repressor and the 56 amino acid N-terminal fragment (Fig. 6). There are differences in the protection patterns among the altered lac repressor peptide sequences tested, which we discuss below.

(d)

(e)

Figure 2. Representative densitometer scans of autoradiograms using a Shimadzu densitometer. The DNA sequence corresponds to the lac operator region being analyzed. It is the left of an almost symmetric sequence centered at + 11. The boxes are sequences that are reflected on the right, beyond + 11. Reproducibility of the peaks’ heights were reproducible to +30%. Footprints are from cells with: (a) intact repressor in HBlOl; (b) intact repressor + TPTG in HBlOl; (c) pTHP64leu61 and pGPl-2 in DH9; (d) pTHP64tyr3leuSl and pGPl-2 in DH9; (e) control with pT7-7 and pGPl-2 in DH9.

(c) Repression of in vivo /?-galactosidase

synthesis

B-Galactosidase assays were performed to test if the DNA interactions, observed by footprinting methods, affect gene expression or transcription at the intended site of action of the la& promoteroperator of the lac operon. This assay uses the host chromosome B-galactosidase gene in the wild-type lac operon of E. coli. The plasmids do not contain enough fl-galactosidase structural gene sequences to yield enzyme activity. Under normal growth conditions, intact lac repressor in the cell represses the production of /?-galactosidase unless the system is induced. In the studies presented here, the ~1857

628

A. M. Khoury

et al.

-u-L:....................

-15

-14

.

-13

-12 -II

-10

-9

-8

-7

-6

-5

-4

-3

-2

-I

I

2

3

4

5

.

6

7

.

8

.

9

*

IO

II

Figure 3. The effects of the wild-type 56 amino acid N-terminal fragment of luc repressor on operator DNA compared with controls in vivo. The nucleotide sequence of the fragment is given at the bottom with the symmetric regions boxed in. We adopted the same method of presentation and estimating levels of methylation as Ogata & Gilbert (1978, 1979). where the change in methylation is given as the logarithm of C/P, which corresponds to the band intensities in the absence (C) and presence (P) of protein. Methylation is enhanced when log (C/P) < 0, reduced when log (C/P) > 0, and unaffected when log (C/P) = 0. C/P values were reproducible to +@3. Data for HP56wt (pTHP56wt and pGPl-2 in DH9, bars with horizontal lines), intact repressor with IPTG (shaded bars) and intact repressor (pT7-7 and pGPl-2 in HBlOl, open bars) obtained in this in vivo study. The DNA sequence is for ZuclJV5 where -9 and -8 have changed from G/C and T/A, respectively. 0.6

o-5

0.4

0.3 & g

0.2

-I 0-I

0.0

-0.1

-0.2 -15

-14

-13

-12

-II

-10

-9

-8

-7

-6

-5

-4

-3

-2

Figure 4. Comparison of 56 and 64 amino acid N-terminal fragment and pGPl-2 in DH9, bars with horizontal lines), HP64wt (pHP64wt (pT7-7 and pGPl-2 in HBlOl, open bars).

-I

I

2

3

4

5

6

7

8

9

IO

II

effects with intact repressor. HP56wt (pTHP56wt, and pGPl-2 in DH9, filled bars), intact repressor

E. coli Repressor

with lac Operator

Interaction

629

0.6

0.5

0.4

0.3

:

0.2

8 -.I 0.1

0.0

-0-I

-0.2 -15 -14

-13

-12

-I I -10

-9

-8

-7

-6

-5

-4

-3

-2

-I

I

2

3

4

5

6

7

8

9

IO

I

Figure 5. Comparison of various 64 amino acid N-terminal fragments’ effects on Zuc operator in wivo. HP64wt (filled bars), HP64tyr3 (open bars), HP641eu61 (shaded bars) and HP64tyr3leu61 (hatched bars) obtained in this study. pGPl-2 in DH9 was used for each with the pTHP plasmid. repressor

is inactivated

to start

transcription

plasmid. The host fl-galactosidase expression is simultaneously induced by addition of 1C3 M-IPTG to remove host genome-produced lac repressor from the operator. Thus, any reduction of fl-galactosidase

at the

T7 polymerase structural gene, which then initiates transcription of the lac repressor gene fragment cloned behind a T7 polymerase promoter of the

0.8

0.6

-0.4:.

-15

-14

. -13

-12

-II

. -10

. -9

. -8

. -7

. -6

. -5

. -4

. -3

. -2

. -I

. I

. 2

. 3

. 4

. 5

. 6

. 7

. 8

. 9

. IO

II

Figure 6. In vitro 0°C data from Ogata & Gilbert (1979) for proteolytically prepared headpiece (bars with horizontal lines), intact repressor (open bars) and repressor +IPTG (shaded bars) shown here for comparison. They used the wildtype operator DNA so the sequenceat -9 and -8 reflect this.

A. M. Khoury

L -Y3

N

-Y3

N

-Y3

et al.

L61 -C

C

C

Lot

repressor

Tetramer

t

tetramer

IPTG

a-Galactosidase

actlwty

Figure 7. Summary of fl-galactosidaseassayresults.The effect of the N-terminal lac repressorfragmentsin the cell are evaluated by measuringthe extent of repressionof the level of b-galactosidaseproduced when host lac repressoris inducedby IPTG. Repressionis measuredas the units of fi-galactosidasein the cell in the presencedivided by units in the absenceof the repressorfragmentsmultiplied by 100.Thesedata representthe averageof 3 to 5 experimentsfor each bar. The assaysfor cells containing N-terminal fragments were done in the presencesof IPTG (seeMaterials and Methods).

in cells containing a pTHP plasmid, when compared with cells treated the same way but containing the control plasmid pT7-7, is due to the cloned N-terminal gene fragments binding to the host wildtype lac operator and interfering with the expression of /?-galactosidase mRNA. Figure 7 shows the effects lac repressor peptide length and amino acid substitutions have on the detected /?-galactosidase activity. It is unlikely that IPTG interacts with the N-terminal fragments since Platt et al. (1973) have shown that the repressor fragment starting with amino acid 100 forms tetramers and has a K, value for IPTG virtually equal to intact repressor. This restart protein is i- in wivo.

4. Discussion Previous in vitro experiments that examine the interaction of proteolytic N-terminal fragments of Zac repressor with Zuc operator (Ogata & Gilbert, 1978, 1979) were contradicted by the report of an inducer-sensitive interaction of lac repressor cores (tetramers missing the N-terminal 51 or 59 amino acid residues) with operator DNA (Matthews, 1979). The experiments demonstrating the lac repressor N-terminal interaction with operator excluded possible artifacts due to contaminating intact lac repressor by using a high concentration of unlabeled operator instead of a control with IPTG present. The experiments to show interaction of the larger

tetrameric “core” with operator (IMatthews, 1979; Manly & Matthews, 1984; Manly et al., 1983, 1984) did not completely rule out the possibility of contamination by tetramers with one, two and/or three intact subunits which interact with the operator (Dunaway & Matthews, 1980). Both sets of experiments were subject to these concerns, since very high levels of Zacrepressor N-terminal fragment (1 to 10 PM) were used in t’he 1:n vitro methylation protection to compensate for the increased 1u.t repressor N-terminal fragment-operator DNA dissociation constant. Thus. even a 1 to So/, con tamination with intact repressor could account for the observed results in either case. The experiments described here confirm that the Eat headpiece alonr can specifically bind the lac operator DNA sequencesin viva. Note that the in U~:VO N-terminal fragment concentrations range from 8 to 80 ,u~ rather than the 10 nM estimate for the intact tetramerit lac repressor concentration normally found in the cell. In addition, the lac operator sequence in these experiments is on a plasmid and therefore it’s concentration is 15 to 20 nM rather than 1 n.v in a cell with only a bacterial chromosome. For comparison, Figure 6 shows the in vitro results that were obtained by Ogata & Gilbert at 0°C for the 59 amino acid Eat headpiece, intact tetrameric lac repressor and intact tetrameric Zac repressor plus IPTG, which are directly comparable to results we obtained in vivo at 4°C. Note that OUI results presented in Figures 3 and 4, although not

E. coli Repressor

Interaction

identical, are similar to the results of Ogata & Gilbert. In both sets of experiments the observations with intact repressor are virtually identical, G protection at + 5, + 7 and + 1 l/12 with enhanced G methylation at +9. The comparison of results with the 56 amino acid N-terminal fragment show substantial protection at the G of + 5 and enhanced methylation at +9 both in vivo and in vitro. Our measurement at +7 for the N-terminal fragment differs with enhancement in vitro seen by Ogata & Gilbert (1979) at 0°C. They do see protection at room temperature, however. Our in vivo results show agreement for the headpiece and the intact protein, with methylation protection at +7 as seen for intact repressor both in vitro and in vivo. This is further direct confirmation that the N-terminal fragments interact at the operator. Similar comparisons of our results and results of Ogata & Gilbert with those of Manly et al. (1983), show good agreement for intact lac repressor G protection at positions +5, + 7 and + 1l/12. The results of Manly et al. (1983) for +9, where protection with the intact repressor controls was observed, differs from our data and the previous work of Ogata & Gilbert where enhancement was seen both in vivo and in vitro. Manly et al. (1983) saw enhancement of + 9 by their “core” preparations. There is less agreement in the comparison of the controls with IPTG in vivo (Fig. 3, shaded bars) and in vitro (Fig. 6, shaded bars). We see enhanced reactivity at +5, + 7 and - 13. This may be a reflection of the transit of RNA polymerase which is absent in the experiments in vitro (Ogata & Gilbert, 1979). An effect at position - 13, seenwith both the intact repressor and all the N-terminal fragments, is possibly due to interaction by RNA polymerase with the DNA at the canonical -10 region that spans 4 bp (Gilbert, 1976; Reznikoff & Abelson, 1980). In the original “footprint” experiment, Schmitz & Galas (1979) showed that E. coli RNA polymerase can appear to simultaneously occupy the -10 region when lac repressor is bound. The result we seemust be due to a differential effect of the presence of the lac repressor fragment. Any effect due to RNA polymerase alone yields a C/P of 1 and is 0 in the log plot (see the legend to Fig. 3). All the cloned N-terminal fragments 64 amino acid residues long protect positions +5, + 7 and + 11 (Fig. 5). Unlike intact repressor they also protect rather than enhance the G residue at +9 from methylation. However, our in vivo, data also show additional interactions at - 2, - 4, -6 and - 13, not normally seen with intact repressor. Figure 4 shows that HP56wt and HP64wt protect G residues in very similar fashion to the intact repressor. The differences are at position + 1l/12, where we see no significant effect with the N-terminal fragments but a slight protection with intact repressor. Like intact repressor the shorter N-terminal fragment HP64wt enhances methylation at +9. The most noticeable difference due to the N-terminal fragments, however, is seen at -6. Comparisons of the various 64 amino acid fragments

with lac Operator

631

with altered sequences in Figure 5 reinforce this observation. Do the changes in the amino acid sequencesat 3 and 61, which result in tighter operator interaction with intact repressor, have an effect on these experiments? HP64tyr3 and HP64tyr31eu61 have similar protection patterns with increased protection at positions + 7, + 9 and + 1l/l2 in both cases,when compared with HP64wt, the normal sequence (Fig. 5). There is also a significant level of protection at -4, - 6 and - 2/- 1 although somewhat more pronounced in the case of HP64tyr31eu61. The increased level of protection at + 7, + 9 and + 1l/12 with these two protein fragments, when compared with the rest of the protein fragments and intact repressor, hints at an increased affinity. Using the same amino acid substitutions in intact repressor, Ogata & Gilbert (1979) reported similar slightly increased protection by the altered proteins with changes in pattern from wild-type protein. The HP641eu61 protection pattern, on the other hand, resemblesthe protection pattern of HP64wt, except for the lack of protection at position -6. This supports the suggestion that the leucine substitution enhances DNA interaction only with the intact tetramer by shifting the quaternary structure to the DNA binding conformation (Ogata & Gilbert, 1979; Maurizot & Grebert, 1988). The biggest overall effect on the methylation pattern was observed with the double amino acid exchange, HP64tyr311eu61. With this protein, in addition to the interactions at positions + 5, + 7 and + 9 (protection not enhancement), there is strong protection at positions -2, - 4, - 6 and - 13. All of these upstream effects were not seen before. These results correlate well with the wholeculture fi-galactosidase assays. Although none of the repressor fragments bind as tightly as, the intact repressor, greater repression is seen with the mutants of the 64 amino acid headpiece. The longer N-terminal fragments HP64tyr3, HP641eu61 and HP64tyr31eu61 inhibit the production of /l-galactosidase similarly and to a larger extent than do HP56wt and HP56tyr3, which are three-fold higher in cellular concentration. The exception, HP64wt represses less than expected from its protection patterns. This is probably due to the fact that it seemsto be the least stable in the cell as shown in Table 1, perhaps due to proteolytic activity. The level of repression is thus directly affected by the lower concentration of protein in the cell. These enzyme assays also exclude artifacts that may be associated with the single lac operator sequenceon a plasmid, since the /?-galactosidase is transcribed from the bacterial chromosome. The new protection sites at - 13, - 6, - 4 and - 2 suggest that the N-terminal peptides can bind sequences similar to the operator, as well as the operator. Interestingly, these sites are centered on sequencesclose to the TGTGAG consensusidentified by Ebright (1985, 1986) as “half” sites for lac repressor-operator interaction. Figure 8 shows the sequence of the entire operator plus some flanking

632

et al.

A. M. Khoury

0.6 o-5 0.4 0.3

,a ; 0.2 J 0-l o-0 -0-I -0.2

4

ul .

-IS-14-13-12-11-10-9-6-7-6

.

-54-3-Z

.

-I

I

2

3 4

5

6 7

8

9 IO II

. . . . . I2 13 14 I5 16 17 I9 19 20 21 22 23 24 2526

. . 27 26

-11-

-2

2-

-3

3Y

-4

95 6 7

Figure 8. Summary of the in wivo methyl&ion pattern of intact Zucrepressorand lac headpiecefrom this work superimposed on publishedin vitro structural data and footprints. Only the top strand wasprobed,and no data is shown beyond base-pair+ 12sincethe next G residueoccursat + 27. From the distribution of operator constitutive mutations, the left half, analyzed here, plays the greater role in Zac operator function (Gilbert et al., 1976; Caruthers, 1980). (1) The filled circles indicate the protein-DNA interaction region from the model describedby Lamerichset al. (1989)derived from ‘H nuclear magneticresonanceresults. (2) (3) (4) Alternate binding sitesfor the headpieceproteins inferred from our data where filled circles are sequence identities with (1). (5) Schmitz $ Galas (1979) footprint of intact top strand. (6) Schmitz & Galas (1979) footprint of intact Zac repressor-bottom strand. (7) Gilbert & fragment protected against DNase by intact repressor transcription followed by RNA polymerase. The the top shows how our data and the model described by Lamerichs et al. (1989) are compatible with N-terminal fragments simultaneously on the Zac promoter-operator region.

Zuc repressor Maxam (1973) cartoon across as many as 4

regions. It illustrates the similarities in sequence at sites adjacent to where the intact repressor functions biologically. The shorter N-terminal fragments appear to interact independently at least with one of these sites between -2 and -6. This suggests that there could be as many as four independently bound N-terminal fragments per operator as shown in Figure 8. This may explain why Buck et al. (1983) found a greater than 4 : 1 stoichiometry for the complex between a 51 amino acid headpiece mixture and a symmetric 62 bp double operator related DNA fragment that includes sequences -5 to +26 twice or potentially six N-terminal binding sites. However, observations reported by Coulard et al. (1982), where fluorescence and circular dichroism monitored titration between 51 amino acid proteolytically prepared 1a.cheadpiece with a 25 bp lac operator DNA, suggested a stoichiometry of 4 peptides to 1 DNA cannot be explained since their DNA is too short. Our observation here that inter-

with a 22 bp operator-related sequence (Lamericks et al., 1989) leads us to suggest that there is space for possibly four N-terminal fragments bound simultaneously. The operator was originally defined by the location of constitutive mutations that occur between positions + 1 and + 17. We cannot exclude the possibility that more than one N-terminal fragment is required for the response seen in Figure 7, or that more than the “operator” sequence is required for repression here. Figure 8 shows this schematically, along with the primary and six similar secondary recognition sequences. Intact. repressor, being a tetramer, must use the 2-fold symmetry of the operator so that only one intact repressor is observed to bind centered on position

action

In this study, two lengths of the lac repressor N-terminal DNA binding domain were successfully

occurs

from

-6

to -2

combined

with

the

recent model for N-terminal fragment interaction

+11.

5. Conclusions

E. coli Repressor

Interaction

cloned and isolated. It was shown that these cloned protein fragments are recognized by polyclonal lac repressor antibodies, yield in vivo footprint patterns similar to intact repressor and repress p-galactosidase synthesis. Altered sequencesin the 64 amino acid lac repressor fragments display more binding and repression, paralleling the same change in phenotype of the intact repressor. The 64 amino acid protein fragment with the single alteration (Pro3 --, Tyr) displays the tightest binding with the center of the operator, G + 5 through G + 11 while the double amino acid exchange, HP64tyr31eu61, shows a stronger methylation protection pattern that spreads from G - 6 through G + 11 on the left half of the symmetric operator. This observation suggests that additional binding sites for the isolated headpiece proteins exist. These data show that N-terminal fragments of lac repressor of varying lengths fold correctly enough to show the expected DNA interaction of the intact repressor. Isolation of these longer fragments will allow ‘H nuclear magnetic resonance structural studies (Zuiderweg et al., 1983; Kaptein et al., 1985; Boelens et al., 1987) residues.

to be extended

beyond

59 amino

acid

We thank J. Betz, M. Brown, S. Tabor and C. Richardson for generous gifts of strains and plasmids. The synthetic DNA used for linkers, primers and mutagenesis were supplied by the University of Pennsylvania DNA Synthesis Service supported by grants from NCI. This work was supported by grants from the NIH to P.L. References Anderson, W. F., Tekeda, Y., Ohlendorf, D. H. 8c Matthews, B. W. (1982). J. Mol. Biol. 159, 745-751. Arndt, K., Nick, H., Boschelli, F., Lu, P. & Sadler, J. (1982). J. Mol. Biol. 161, 439457. Betz, J. $ Sadler, J. R. (1978). Gene, 3, 26%278. Boelens, R., Scheek, R. M., Lamerichs, R. M. J. N., de Vlieg, J., van Boom, J. H. t Kaptein, R. (1987). In DNA-&and Interactions (Guschlbauer, W. & Saenger, W., eds), pp. 191-215, Plenum, New York. Bowie, J. U. & Sauer, R. T. (1989). J. Biol. Chem. 264, 7596-7602. Brown, M., Figge, J., Hansen, U., Wright, C., Jaeng, K.-T., Khoury, G., Livingston, D. & Roberts, T. (1987). Cell, 49, 603-612. Buck, F., Hahn, K.-D., Zemann, W., Riiterjans, H., Sadler, J. R., Beyreuther, K., Kaptein, R., Scheek, R. & Hull, W. E. (1983) Eur. J. Biochem. 132, 321-327. Caruthers, M. (1980). Act. Chem. Res. 13, 155-160. Church, G. M. & Gilbert, W. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 1991-1995. Coulard, F., Schnarr, M. & Maurizot, J. C. (1982). EMBO J. 1, 14051409. Davis, L. G., Dibner, M. D. $ Battey, J. F. (1986). In Basic Methods in Molecular Biology, pp. 233-237. Elsevier Science Publishing Co., New York. Dunaway, M. & Matthews, K. (1980). J. Biol. Chem. 255, 1012610127. Ebright, R. H. (1985). J. Biowwl. Struct. Dynam. 3, 281-297. Ebright, R. H. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 303-307.

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by P. von Hippel