J. Mol. Biol. (1991) 218, 313-321

The Roles of Residues 5 and 9 of the Recognition of Lac Repressor in Zac Operator Binding

Helix

Jiirgen Sartorius, Norbert Lehming, Brigitte Kisters-Woike Brigitte von Wilcken-Bergmann and Benno Miller-Hill Institut fiir Genetik der Universitcit xu Kdn Weyertal 121, 5000 Kdn 41, Germany (Received 10 August

1990; accepted 28 November 1990)

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts nonspecifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.

1. Introduction

exchanges. We have used such an experimental approach to analyse recognition between lac operator and Lac repressor. We have described a system of two compatible plasmids that can be used to characterize proteinDNA interactions in viva in E. coli (Lehming et al.; 1987). The plasmid pWB300 carries the la& reporter gene under the control of a wild-type lac promoter in which the natural first lac operator has been replaced by a unique XbaI site for the insertion of synthetic lac operator variants. The other plasmid, pWB1000, contains a semi-synthetic wildtype lad gene with conveniently positioned unique restriction sites. For a more detailed description, see Lehming et al. (1987). We used this system to introduce predetermined amino acid exchanges into the recognition helix of Lac repressor. The affinities of the resulting Lac repressor mutants were then tested for a variety of symmetrically substituted variants of the ideal lac operator in vivo and in vitro (Lehming et al., 1987, 1988; Sartorius et aZ.. 1989). We have also constructed several expression libraries for Lac repressor mutants with all the possible amino acids at distinct positions of the recognition helix and screened these for their ability to recognize particular operator variants. The combined efforts have yielded a large collection of Lac repressor mutants that bind very well to individual lac operator variants. Thus, we have been able to determine the orientation of the recognition helix of Lac repressor with respect to the centre of symmetry of lac operator (Lehming et al., 1988) and to deduce rules for the interactions of residues 1 and 2 of the recognition

The helix-turn-helix motif is a prominent structure used by many DNA-binding proteins to recognize their specific targets. The C-terminal cl-helix of this motif, the so-called recognition helix, crosses the major groove of B-DNA and is held in place by protein-protein interactions with the N-terminal u-helix. Amino acid side-chains of the recognition helix that face the DNA recognize specific structures of the major groove of the operator sequences. Evidence for the function of the helix-turn-helix motif has been provided by X-ray analyses of viral repressor-operator complexes (Anderson et al., 1987; Aggarwal et al., 1988; Jordan & Pabo, 1988; Wolberger et al., 1988). The existence of a helixturn-helix motif in the Escherichia coli Lac repressor was deduced first from sequence comparisons (Matthews et al., 1982) and then confirmed by NOEt measurements in H n.m.r. spectra of the Lac headpiece-lac operator complex repressor (Lamerichs et al., 1989). The interactions between amino acids and basepairs have also been characterized by genetic methods: for the CAP protein (Ebright et al., 1984), and the 434 cl repressor (Wharton & Ptashne, 1987) mutants have been described with amino acid substitutions in t’he recognit’ion helices that no longer recognize their nat’ural targets but, instead, bind to altered targets with particular base-pair 7 Abbreviations used: KOE, nuclear Overhauser enhancement; n.m.r., nuclear magnetic resonance; IPTG, isol”opYl-8-n-thiogalactoside. 313 002%2836/91/060313-09

$03.00/O

0

1991 Academic

Press Limited

J. Sartorius

314

helix, corresponding to ‘residues 17 and 18 of Lac repressor, with base-pairs 4 and 5 of the operator (Lehming et aE., 1990). We have demonstrated further that residue 6 of the recognition helix, corresponding to residue 22 of Lac repressor, interacts with base-pair 6 of the operator in a manner that is independent of the nature of the interactions between residue 2 and base-pair 4 (Sartorius et al., 1989).

The inspection of a helical wheel projection of the recognition helix shows that the amino acids in positions 1, 2, 5, 6 and 9 are directed towards the DNA. We thus attempted to analyse the contributions of the remaining residues 5 and 9 of the recognition helix, corresponding to residues 21 and 25 of Lac repressor, to operator recognition. For this three different Lac purpose we constructed repressor expression libraries. With the help of one of these we are able to demonstrate that residue 5 participates in specific repressor-operator interactions. Results obtained with the other two mini libraries together with model building studies suggest that residue 9 of the recognition helix influences operator binding by interaction with the DKA backbone.

2. Materials (a) Media, strains,

and Methods

chemicals a.nd general methods

All media, chemicals and general methods used were the same as described by Lehming et al. (1987). All oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer and purified on denaturing polyacrylamide gels before cloning. Strain DH5a, used for the analysis of the libraries, has the genotype F-

endA hsdA17 (rk-, relA a180 dZac2 hMl5.

mk+)

supE44 thil 1~’ recA gyrA96

Strain DC41-2, used for determination of specific P-galactosidase activities by the method of Miller (1972), has the genotype (Zac pro), galE smR recA. (b) The y WBl125

library

By offering a mixture of cytosine and guanine only for incorporation at the 3rd positions of codons 1, 2 and 5 of the recognition helix we reduced the respective number of possible codons by a factor of 2 and the complexity of the library by a factor of 8 (see below). Other advantages of this procedure are the exclusion of 2 stop eodons (UAS and UGA) and the 2-fold enrichment of the codons for methionine and tryptophan. In order to avoid any serine codons in position 5 of the recognition helix we synthesized 3 oligonucleotides (Fig. l(a)): in oligonucleotide A we offered only cytosine and guanine for position 1 and a mixture of all 4 nucleotides for the 2nd position. Codons beginning with adenine or thymine were generated in separate reactions so that in the case of adenine in posit(ion 1 (oligonucleotide B) guanine was excluded from position 2 and in the case of thymine in position 1 (oligonuleotide C) no cytosine could be incorporated in position 2. Owing to this arrangement codons AGC, TCC and TCG for serine and 1 of the eodons for arginine (AGG) are missing in position 5 of the recognition helix. The calculated complexity of the pWBl125 library thus amounts to 32 codons x 32 codons x 28 codons = 3 x IO4 codons. which is about 1 order of magnitude

et al

below the complexity completely randomized

of a corresponding library with 3 codans (64 x 64 x 58 z 2.3 x 105).

(c) The His1 Gln2 Ala5 X9 and

Vail

Ala2 Ser5 X9

mini-libraries These mini-libraries were constructed with a synthetic oligonucleotide coding for amino acids 25 to 34 of the Lac repressor headpiece (residue 25 corresponds to residue 9 of the recognition helix) (Fig. l(b)). This oligonucleotide was designed to carry 32 different codons in position 25 in the same manner as described for oligonucleotides A, B and C of the pWB1125 library. The procedures of filling in and (Fig. l(a)) except digestion were done as for pWB1125 that HpaI, which cleaves between amino acids 8 and 9 of the Lac repressor recognition helix, was used in place of NaeI. The synthetic fragment was then cloned between the proper sites of 2 different pWB1000 derivatives whose lad genes code for the His1 Gln2 Ala5 or t,he Vail Ala2 Ser5 repressor mutant, respectively. The Lac repressor mutants are named according to the amino acids they carry in positions 1. 2 and 5 of their recognition helices. Since the calcula,ted complexity of both mini-libraries was 32 (4 x 4 x 2) we were sure to see every possible amino acid in position 9 if we screened 5 x IO* clones in combinntion with every lac operator variant indicated in ResuIts. The screening procedure was the same as used for the pWB1125 library. (d) A simpl;fied

Screen of /&galactosidase

activity

A 0.5 ml sample of an overnight culture in Yl’ medium supplemented with the respective antibiotics and with or without 5 x 1O-4 w-IPTG was spun down in an Eppendorf tube. The cells were resuspended with 0.5 ml of Z-buffer (Miller, 1972) and then disrupted with 10 ,uI of toluol for 1 min. The reaction was started by addition of IO0 ,nl of 4 mg OrVPG (o-nit,rophenyl-fi-n-galactoside)/ml and stopped with 250 ,a1 of 1 tir-ru‘a,CO, after 5 min. The intensity of the yellow colour was judged visually by comparison wit’h a constitut,ive standard. Clones that yielded reaction mixt~ures of less-intense colour when grown in the absence of IPTG but resembled the constitutive controi when grown in the presence of IPTG were analysed further. This method is much more ra,pid but less exact than the determination of the specific j?-galaetosidase activity by the method of Miller (1972); and so it was onlyused as a quick qualitative assay in order to choose the clones w-ith the lowest but inducible j-galactosidase activity for further characterization.

(e) #/lodeI building as,d cabxlations lModel building was done on an Evans and Sut~herlantl PS330 graphic system hosted by a MicroVax II with INSTGHT. Calc&tions were done on a Gray X/MP-48 with DISCOVER (INSIGHT and DISCOVER were from Biosym Inc., Sa,n Diego, CA). 3.

(a)

Construction

In order

Results

of the Lx repressor p WBl I%3 expression library

to examine

the

role

of the amino

acid

in

position

5 of the recognit.ion helix of l,ac repressor in the binding t,o lac operat)or we constructed the pWR1125 expression libra~ry (Fig. l(a)). The clones contained

in

pVVBfl2.5

should

express

all

the

Protein-DNA

Recognition

315

Codcns

25 to

of

34

the

1x1

(repressor)

gene

AA 5, ccc GGG

AA

AA

B s’-

cccccc

c5,-

AA AA cccccc GGGGGG-TG TT TT

CGGGGGTT TT

AC

AC TG

*nneot

TG

with

coding

for helix:

with

primer

AC

~igote

Liqote

3’

NoeI/XmQI-res?ricted

n,,.I/XmoILac Vail

restricted

repressors Ala2

SerS

mutant or

His1

pWBlOO0 in the Gln2

DNP.

recognition

Ala5

pWBlOOOONA

Figure 1. (a) Construction of the Lac repressor pWB1125 expression library. A, B and C are schematic representations of synthetic oligonucleotides. Each consists of the 87 bases of the ZucI gene from codon 10 to codon 39. They are identical except for codon 5 of the Lac repressor recognition helix (serine 21 of Lac repressor). The codons 1 and 2 (tyrosine 17 and glutamine 18) are randomized so that all possible amino acids may be incorporated at these positions. The use of a mixture of guanine and cytosine only at the 3rd position reduces the overall complexity of this library, excludes the stop codons UAA and UGA and raises the relative frequency of the single codons for methionine and tryptophan (see also Materials and Methods). Three independent oligonucleotides were synthesized in order to avoid any serine codon at position 5 of the recognition helix. Since sample A allows 16 different codons at this position and samples B and C 6 codons each, they were mixed at a ratio of A : B : C = 8 : 3 : 3 prior to annealing to an 18 base primer. The products of fill-in reactions with Tap polymerase in the presence of an excess of all 4 nucleotides were extensively digested with NaeI and XmaI. The resulting DNA fragment was ligated between the NaeI and XmaI sites of pWB1000. (b) Construction of the Lac repressor mini-libraries, His1 Gln2 Ala5 X9 and Vall Ala2 Ser5 X9. A DNA fragment was synthesized that codes for the amino acids 25 (=residue 9 of the recognition helix) to 34 of the Lac repressor. During the synthesis of this oligonucleotide all 4 nucleotides in the 1st and 2nd position and only C and G in the 3rd position of codon 25 were offered, to allow 32 possible codons. After annealing to an 18 base primer a fill-in reaction with Tap polymerase in the presence of an excess of all 4 nucleotides creates a double-stranded DNA fragment with a blunt 5’ end. This was digested with XmuI and ligated between the blunt HpuI site and the sticky XmuI site of the pWBlOO0 derivatives coding for the His1 Gln2 Ala5 and the Vall Ala2 Ser5 repressor mutants, respectively. bp, base-pair; b, base; ds, double-stranded.

possible amino acids in positions 1, 2 and 5 of the recognition helix of Lac repressor in all possible combinations, with one exception: serine should be excluded from position 5. From earlier results obtained with a library variable only at positions 2 and 5 of the recognition helix (data not shown) and from X-ray analyses of the repressor and Cro protein of phage 434 (Mondragon et al., 1989a,b), we concluded that the function of amino acid 5 depended largely on its ability to interact with the amino acids in positions 1 and 2 of the recognition helix. We designed a mixture of oligonucleotides in such a way that the six possible codons for the wild-type amino acid serine were avoided in position 5 of the recognition helix (Fig. l(a)). We did this to avoid re-isolating those repressor mutants with amino

acid exchanges only in positions 1 and 2 of the recognition helix, which bind tightly to several different operator variants (Lehming et al., 1987, 1990; Sartorius et al., 1989). The oligonucleotides were inserted into the sequence coding for the lad gene in pWBlOO0 at the proper position to yield the pWB1125 library (see Fig. l(a)). We obtained about lo5 independent clones. Since we offered only guanine and cytosine for the third “wobble” base-pair of the variable codons and avoided the codons for serine in position 5 (Fig. l(a)), we expected to have about 3 x lo4 (7=,rl;:2 x 28) different DNA sequences encoding protein (=20X20X 19) different sequences. To exclude the possibility of cloning artefacts we used a sample of the amplified pWB1125 library for sequence analysis (Sanger et

J. Sartorius

316

al.; 1977). We could read the sequence clearly from the first 50 eodons of Lac repressor except for the codons in positions 1, 2 and 5 of the recognition helix, where bands of roughly equal intensities appeared in all four tracks or in the G and C tracks, respectively (data not shown). Thus, we were confident that we would be able to detect all functional Lac repressor mutants in this library, which was then used to transform E. coli cells harbouring a compatible plasmid, a derivative of pWB300 with the lacZ reporter gene under the control of one of our lac operator variants with symmetric exchanges at base-pairs 3, 4 and 5 (Fig. 2). (b) Analysis

of the Lac repressor p WB1125 expression library

A 10 ng sample of pWB1125 DNA was transformed into competent cells of the E. coli strain DH5a, harbouring a pWB300 derivative, with the ideal symmetrical lac operat,or or one of its symmetrical variants altered in base-pairs 3: 4 and/or 5 (Fig. 2). We chose these operator variants for the analysis of pWB1125 because we expected interactions of amino acid 5 with the operator DNA in the region between base-pair 3 and base-pair 5. 1acZ gene in such a way DH~E carries an inactive that the measurable /I-galactosidase activity originates from the la& gene under the control of the respective lac operator variant. Cells carrying the two plasmids were screened on minimal agar plates containing ampicillin, tetracycline and 5-bromo4-chloro-3-indolyl-/?-n-galaetoside (Xgal). Colonies in which Lac repressor mutants bind to the respective lac operator variants and thereby repress the expression of /I-galactosidase appear a lighter blue on these indicator plates compared with those in which P-galactosidase is expressed constitutively. Light blue colonies were subjected to a simplified screen for /I-galactosidase activity (see Materials and Methods) and the lightest blue clones were tested for their inducibility by TPTG. Plasmid DNA was prepared from inducible clones and t’he regions coding for the headpiece were sequenced according to the method of Sanger et al. (1977). Simultaneously, these DNA preparations were incubated with the endonuclease ClaI, which cuts the plasmid pWB300, which carries the 1acZ gene but leaves the pWB1125 derivative intact. Transformation with the ClaI-restricted DNA yields transformants that are resistant to ampicillin, but no transformants resistant to tetracycline. By this procedure we separated the lad plasmid from the 1acZ plasmid in order to ensure that the repressed phenotype observed in the first screen did not result from an alteration in the 1acZ gene or the host bacteria. The CEaI-restricted plasmid DNA from the lightest blue colonies was then transformed into cells of the E. coli strain DC41-2 harbouring the pWB300 derivatives with the operator variants of interest. The specific activity of /?-galactosidase was then determined according to the method of Miller (1972).

et al.

PWB

Iac

umQressed

operator

987654321 310

AATTGTGAGC

GCTCACAATT

4400

TTAACACTCG*CGAGTGTTA

23456789 G C A

5800 13000 5300

AC

GT

AG

CT

3600 4200 5300

332 333 334

C G T

332-41 333-41 334-41

AT

AT

332-42 333-42 334-42

cc

GG

CG

CG

CT

AG

332-44 333-44 334-44

TC

GA

TG

CA AA

3400 4000 2400

T G P.

4500 4000

TT

341 342 344

A C T

3000 3500 3000

3800

7400 4200 4000

341-51 341-52 341-53

AA

TT

CA

TG

GA

TC

342-51 342-52 342-53

AC

GT

5500

cc GC

GG

5100 5700

344-51 344-52 344-53 351 352 353

AT

CT GT

GC AT AC AC

7300 3900 5800

A

T

5900

C

G

G

C

7000 3600

15 2 4

Figure 2. Lac repressor mutants found in the pWBlld.5 expression library. The ba,se-pairs of the idea,] syrnmetrical lac operator (310) are numbered in 3’ to 5’ direction from the eentre of symmetry. Base-pairs 3. 4 and 5 were symmetrically exrhanged to create the operator variants. The first number of a variant stands for the position of the base. The second number is 1 for adenine, 2 for cytosine, 3 for guanine and 4 for thymine. Thus, in the operator variant 41. base 4 is A. 341 designates a p,\VBYOO derivat,ive carrying operator variant 41. The specific activity of /3-galactosidase is obtained from the la& gene under the control of the respective kc operator variant. All these values of specific activity are determined according to the method of Miller (1972) in the presence of the Lac repressor deletion mutant Al. In this mutant the major part, of the headpiece, including the recognition helix (amino acids 14 to 60 of the Lac repressor), have been deleted. Thus, any repression is excluded. Note that no Lac repressor mutant was found with det,ectable affinity to any lac operator variant substituted in base-pair 3.

With each operator variant shown in Figure 2 we examined 40,000 to 50,000 individual colonies of pWB300 harbouring DH& cells transformed with pWB1125. When we screened the operator variants with symmetric substitutions in base-pair 3 or in base-pairs 3 and 4 we found no repressed clones. with With the ideal lac operator and the variants symmetric substitutions in base-pairs 4 and/or 5 we found 37 different- repressing Lac repressor mutants altogether that recognize one or several opera,tor

Protein-DNA variants. Many repressor mutants were isolated several times but with different codons for the same amino acid. These results suggest that amino acid 5 of the recognition helix interacts with base-pairs 4 and 5 of the operator and that base-pair 3 does not participate in the interactions (Fig. 2). Thus, we could omit operator variants with exchanges in base-pair 3 from further analysis and concentrate on the lac operator variants altered in base-pairs 4 and/or 5 (Fig. 3).

352 353

Recognition

317

(c) Residue 5 of the recognition helix interacts with base-pair 4 of the operator The mutant Lac repressors and their repression values obtained with the lac operator variants in base-pairs 4 and/or 5 are shown in Figure 3. The repressor mutants are grouped according to amino acid 5 and within each group according to amino acid 2 of the recognition helix. Five different residues were found in position 5: Met, Gly, Thr, His and Ala. Met5 is found mainly when Thr, Met or Ser are present in position 2 or, apart from that, when

c G

342-51 342-52 342-53 342

AC cc GC c

344-51 344-52 344-53 344

AT CT GT T

342-5, 342-52 342-53 342 344-51 344.52 344.53 344

Figure 3. Repression values of the repressor mutants from the pWB1125 library. The mutant repressors were tested with all the lac operator variants substituted in base-pairs 4 and/or 5 (compare with Fig. 2). Repression is defined as the specific activity of fi-galactosidase in the presence of the Lac repressor deletion mutant Al (legend to Fig. 2) divided by the specific activity of /I-galactosidase in the presence of the respective Lac repressor mutant. Unavoidable rare loss (0.5%) of EacI plasmids carrying an ampicillin resistance gene limits repression to a value of 200. The ZucZ gene is under the control of the operator variant indicated (for further details, see Lehming et al., 1987). The Lac repressor mutants are named according to the amino acids they carry in positions 1, 2 and 5 of their recognition helices. The usual l-letter code is used for amino acids. The mutants His1 Thr2 Gly5 and His1 Ala2 His5 do not originate from the pWB1125 library but have been constructed for comparison. ru’ote that some Lac repressor mutants achieve significant repression with more than one lac operator variant. wt, wild-type.

1. Sartoriw

318

et af. probably not found because serine was excluded from posit,ion 5. Gln, the wild-type amino acid in position 2 of the Lac repressor recognition helix was found t,wice, in the mutants Glyl Gln2 Met5 and His1 Gln2 Alas. Most of the repression values obtained with ang; of the Lac repressor mutants are lower than those obtained with the wild-type Lac repressor. When comparing the repression values of some of the new mutants with glycine in position 5 with the corresponding mutants that carry the same amino acid substitutions in positions 1 and 2, but have t)he wild-type serine in poskion 5, we made a striking observation (Fig. 4). Except’ for the repressor mutant His1 Thr2 Gly5, all the mutants with Giy.5 and Ala2 or Thr2 have lost their capacity to reeognize t’he operator variants with an adenine in position 4 (341-51, 341-52, 341-53 and 341; see legend to Fig. 2 for an explanation of va,riant numbering).

Gly is present in position 1, Gly5 occurs only together with Ala or Thr in position 2 with only one exception: the mutant Serl Ser2 Gly5. Thr5 is often found with Ala in position 2. Net in position 2 may be combined with Thr5 in the same recognition helix only when residue 1 is His or Arg,. Ala5 also is only present together with His or Arg m position 1. His5 occurs only together with Hisl. These findings hint at some interaction between the amino acid in position 5 and those in positions 1 and 2. A given residue in position 5 will tolerate only a very small number of residues in position 2 of the recognition helix, Otherwise, the binding to any of the examined operator variants will be severely impeded. There are more possibilities of substituting residue 1 whenever there is Met, Gly or Thr in position 5, and His or Ala in position 5 seem to require His or 4rg in position 1. Furthermore, the wild-type amino acid in position 1, Tyr, was most

Ia.2 operator Repression voIues obtolned with LOC repreSSOr mulOnfS SlA2G5 AlA2GS Vl AZG5 11A2G5 TlA2G5 SlT2GS PlTZGS ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

"lTZG5

H1TZH5

H1QZA5

YlQ2S5 OPC

987654321 id

310

AATTGTGAGC*GCTCACAATT

37

9 I51

29 ,751

10 ,401

115 125)

>200 125)

14 (13)

30 ,621

28 17,

16 (7)

A

,241

A

13 (71

15 (7)

61

15 (61

1 11 I

,561

I:,

7 (6)

(:I

40 ,101

2,

351

A

T

33 (7)

352

c

G

1 (21

353

c

G

6,

341-51

341-52

AA

CA

TT

1 ,701

GA

342.51

342-52

342-53

342

344-51

344-52

344-53

A

AC

cc

GC

c

AT

CT

GT

(20)

TC (5:)

341

I

G

AT

,,A,

1 (11,

(1401

4

,225)

3 I1 001

T

3

4

14

1 !2,

(3;)

ill

1 (1 I

A

I:,

u:,

/ I1 1

1 Cl)

1 II,

(2,

12 (60,

II

1 i? ,

6,

5 12,

(3:)

I1 I

2

2 I

4

3 ,801

AG

1

A (245)

I

12

(1)

3 ,:'o,

,:b,

,A 3

1 (3)

AC

!l

4

5

T

,L2& 344

10

4

GG

GC

27 (20)

wb,

A

T

GT

15 ,:I

28 118,

it20Zo

28

8 (23)

1

TG

u:, 341-53

2 (401

clzab,

3 151

2200

150 @ZOO,

(7,

A 8

27 124)

,::,

63 120)

I%

(82, 120 I2200

7 I

Figure 4. Comparison of repression values. Some repressor mutants found in the library pWB1125 (see Fig. 3) are compared with corresponding repressor mutants that carry the same amino acid exchanges in positions 1 and 2 (residues 17 and 18 of Lac repressor), respectively, but the wild-type residue serine in position 5 (residue 21) of the recognition helix. Some of the repressor mutants with wild-type serine in position 5 of the recognition helix have been published: His1 Gln2 Ser5, Vail Ala2 Ser5 and Ala1 Ala2 Ser5 (by Lehming et al., 1987); Serl Ala2 SerS, Ilel Ala2 Ser5, Serl Thr2 Ser5 and Pro1 Thr2 Ser5 (by Sartorius et al., 1989). The others are newly constructed for comparison. The repressor mutants Serl Ala2 SerS, Ala1 Ala2 SerS, Serl Thr2 Gly5 and Pro1 Thr2 Gly5 are active dimeric Lac repressors each encoded by a ZacI gene with a frameshift mutation in codon 330 (Lehming et al.; 1988; Oehler et al., 1990). Comparisons of repression values obtained with dimeric and tetrameric Lac repressors indicate that they never differ by more than a factor of 2 (Lehming et al., 1988). Repression values in parentheses belong to the Ser5 mutants, which are also shown in parentheses at the top.

Protein-DNA

In contrast, their affinity for operator variants with the wild-type guanine in position 4 (351, 352, 353 and 310) is barely affected. Repression of these lac operator variants is more or less of the same order of magnitude irrespective of whether the mutants carry a Gly or a Ser in position 5 of the recognition helix. Thus, the recognition of an A.T pair in position 4 depends on serine in position 5. Binding to the lac operator variants with a T. A pair in position 4 (344-51, 344-52, 344-53 and 344) is also mostly reduced when Ser5 is exchanged for Gly, but to a lesser extent than with the operator variants that carry an adenine in position 4. These general tendencies seem to be largely independent of the nature of the base-pair in position 5 of the operator. The last three repressor mutant,s (His1 Thr2 Gly5, His1 ThrZ His5 and His1 Gln2 Ala5) were included in Figure 4 because of their high repression values obtained with the ideal lac operator (310) and the operator variant 344. The triple mutant His1 Thr2 His5 recognizes six operator variants better than the corresponding double mutant His1 Thr2 Ser5.

9 of the recognition helix seems to bind to the phosphate backbone of the operator

(d) Residue

Computer modelling studies (data not shown) suggested that the asparagine in position 9 of the Lac repressor recognition helix could interact with the backbone of the lac operator DNA, most probably with the phosphate between base-pairs 3 and 2. As a less likely alternative the binding to base-pair 3 of the operator was considered. In order to test the second possibility, we constructed two Lac repressor mini expression libraries that contained all the possible amino acids in position 9. For one of these libraries we used the Lac repressor mutant His1 Gln2 Ala5 to construct the His1 Gln2 Ala5 X9 library from a mixture of 32 (see Materials and oligonucleotides different Methods for details). We obtained about 1 x lo4 independent clones. Since the His1 Gln2 Ala5 mutant recognizes the operator variant 344 (see Figs 3 and 4) with a repression value of 120, we chose the operator variants 344, 344-32, 344-33 and 344-34 for an analysis similar to that described for the pWB1125 library. With each operator variant we screened about 5 x lo2 colonies transformed with the Lac repressor His1 Gln2 Ala5 X9 library. The only repressing mutant that could be detected in this library was the His1 Gln2 Ala5 Asn9 repressor binding to operator variant 344. This mutant hardly binds to the operator variants 344-32, 344-33 and 344-34 as demonstrated by the repression values of 1, 2 and 4, respectively. In the second approach we constructed a similar Lac repressor expression library with Vall Ala2 Ser5 and all the possible amino acids in position 9. The analysis was done in the same manner except that we used the lac operator variants 341, 341-32, 341-33 and 341-34 with an adenine in position 4, because adenine is very well recognized by the Vall

Recognition

319

Ala2 Ser5 mutant (see Fig. 4). Again we failed to detect any repressing Lac repressor mutants except for the initial Vall Ala2 Ser5 Asn9 repressor, which was isolated several times. This is particularly striking, given that the repression values of this mutant obtained with the operator variants 341-32, 341-33 and 341-34 are low. Even a modest binding capacity of any other residue in position 9 should have been easily detected. From these results we propose that the amino acid in position 9 of the recognition helix most probably interacts with the backbone phosphate (Fig. 5).

4. Discussion (a)

The function

of residue

5

of the recognition

helix

In previous experiments we have identified specific interactions between residue 2 of the Lac repressor recognition helix and base-pair 4 of the lac operator, and between residue 6 and base-pair 6 (Lehming et al., 1987, 1988; Sartorius et al., 1989). Unambiguous evidence for these interactions was provided by striking specificity changes. We then exchanged the serine in position 5 of the recognition helix for some other amino acids, but none of these significantly recognizes any operator variant (data not shown). On the other hand, the calculated factors for residues 1 and 2 (Lehming et al., 1990) strongly suggested a contribution to specific binding by at least one more residue. This discrepancy prompted us to construct and analyse Lac repressor libraries with simultaneous random amino acid exchanges in several positions

9R7654321

1

RTTGTGilGCGCTCtlCIlAT 3’

5’

5’

3’

TAACflCTCGCGR-GTGTTA 123456789

Figure 5. Schematic representation of the interactions helix of Lac of residues 2, 5, 6 and 9 of the recognition repressor (corresponding to amino acids 18, 21, 22, 25) with an ideal symmetrical lac operator. In this scheme the helix-turn-helix motif is behind the operator DNA. The large arrow points to the centre of symmetry of Zac operator. Numbering of the bases is as in Fig. 2. The helix-turn-helix motifs are shown in their approximate positions across the major groove as deduced from NOE data (Lamerichs et al., 1989), our genetic analyses (Lehming et al., 1987, 1988; Sartorius et al., 1989) and from model building data (Kisters-Woike et al., unpublished results). N marks the N terminus of helix I. The presumptive interactions of residues 2, 5, 6 and 9 of the wild-type Lac repressor recognition helix are indicated by arrowheads and small arrows.

320

J. Sartorius

of the recognition helix. Only when we permitted a choice of residues 1 and 2 were we able to identify substitutions at position 5 with considerable affinity for particu1a.r operator variants. The data in Figures 3 and 4 show that residue 5 of the recognition helix may help to establish specific contacts to base-pair 4 in co-operation with residue 2. Residue 1 seems to be less involved, except that the spatial restrictions imposed by the wild-type tyrosine prohibit the slight adjustments that seem to be necessa,ry for the establishment of new specific contacts (KistersWoike et aZ., unpublished results). This is also illustrated by the failure to find any repressor mutant with a new specificity in a library with random amino acids in positions 5 and 2 only (data not shown). In Figure 4 we compare Lac repressor mutants that have alanine or threonine in position 2 and glycine in position 5 with the respective mutants that have serine in position 5 of the recognition helix. It can be seen that the Ser5 mutants are less specific than the Gly5 mutants, which recognize operator variants with a G. C pair in position 4 better than the others. The Ser5 mutants prefer an A. T, but, in addition, they recognize G. C or T. A pairs fairly well. Thus, in the case of the mutants shown in Figure 4, recognition is narrowed by the introduction of glycine in position 5 of the recognition helix. We examined a computer model of the Lac helixturn-helix motif (Kisters-Woike et al., unpublished results) for a possible explanation of the experimental results. This model was constructed on the basis of n.m.r. measurements (Boelens et aE., 1988) and our genetic data. It consists of the wild-type repressor docked to an ideal lac operator. At first we exchanged Ser5 for glycine. After slight modifications of the 0 and Y angles (Anfinsen & Scheraga, 1975) for glycine we were able to introduce a hydrogen bond between the backbone oxygen atom of glycine and the amino group of a cytosine in position 4. The helical structure, including the pattern of hydrogen bonds, was not disturbed by this slight adaptation. The newly introduced contact may be established only with Gly5, without distortions of the surrounding repressor-operator contacts, because the appropriate CD and Y angles are only accessible in the glycine configuration space. A. T or G. C pairs will, according to this model, not be recognized by Gly5 mutants, since they lack an amino group in the appropriate position. A T. A pair may also be recognized by glycine in position 5 but less well than G’ C. Serine in position 5 of the recognition helix may act by different mechanisms. Its methyl group contributes to the hydrophobic surface and its hydroxyl group may take part in dipole-dipole interactions or hydrogen bonds with oxygen 4 of the thymine in position 4 of the operator variant 341-51. Taken together, the data of Figures 3 and 4 indicate some influence of residue 5 on the strength of the interaction between residue 2 of the recognition helix and base-pairs 4 and 5 of the lac operator.

et al. But there is no firm evidence that the wild-type serine in position 5 makes direct specific conbets to any base-pair, The few amino acids that’ also function in this position of the recognition helix seem to co-operate with residue 1 in reducing or expanding the space available for residue 2. The possibilities for residue 2 to contact various base-pairs are thereby determined. Only glycine (see above) and hist’idine in position 5 may be mvolved in direct contacts: the His1 Thr2 His5 repressor recognizes the ideal operator at least ten times better t’han the His1 Ths2 Ser5 or the His1 Thr2 Thr5 mutant and twice as well as the His1 Thr2 Gly5 repressor (Figs 3 and 4). This might be taken as an indication that Hi&, indeed, makes direct specific contacts t,o base-pairs 4 and/ or 5 while the other amino acids that, have been examined do not. (b) The function

of residue 9 of the recognition

helix

Reports about homeo domain proteins (Hanes & Brent, 1989; Treisman et al., 1989) suggested a closer look at residue 9 (Asn25) of the recognition helix of the Lac repressor. In the cases of the Drosophila proteins paired and fushi taram, an exchange of residue 9 of the respective recognition helices was sufficient to switch their specificities, although the two target, sequences differ in several base-pairs. Very recently, the structure of t’he antennapedia homeo domain eomplexed with target DPiA has been determined by n.m.r. spect,roscopy (Otting et al., 1990). Comparison of this complex with the 434 repressor operator complex (Aggarwal et ai., 1988) indicates that the helix-turn-helix motifs of bacterial and phage repressors and of homeo domain proteins bind with different segments of their recognition helices to their respective targets (Otting et al.; 1990). 1” Repressor and 434 repressor both carry an asparagine in position 9 of the recognition helix. In both cases X-ray crystallographic analyses (Jordan & Pabo, 1988; Aggarwal et al., 1988) have shown that residues 9 (i.e. Asn52 of 1” repressor and Asn36 of 434 repressor) contact the phosphates between A7 and T8 (numbering of base-pairs is from the eentre towards the outside and zero is assigned to the central base-pair of ;1 operator). Model building with the Lac repressor helixturn-helix motif and lac operator D?;A (KistersWoike et al., unpublished results) suggested that the side-chain of Asn25, the last residue of the recognition helix, could come close to base-pair 3. A contact between the y amino group and the phosphate t,hat connects C and T in positions 2 and 3 on the lower strand (Fig. 5) was likely but additional interactions, i.e. specific recognition of base-pair 3; could not be excluded by model building. Operator variants with base-pair exchanges in position 3 are poorly recognized by wild-type Lac repressor. The values of in viz10 repression obtained with the lac operator variants 332, 333 and 334 are 2, 5 and 2, respectively (Lehming et al., 1987). This strong reduction of affinity seems to indicate specific recognition of base-pair 3. The results

Protein-DNA

obtained with the pWB1125 library exclude the recognition of base-pair 3 by residue 5 of the recognition helix. In order to test for a possible contribution of residue 9 of the recognition helix (Asn25) to specific recognition we used the His1 Gln2 Ala5 and the Vail Ala2 Ser5 mutants as background for X9 libraries because they bind tightly to the operator variants 5’-AATTGTTAGC: (344) and 5’-AATTGTAAGC: (341), respectively. In both cases binding is drastically reduced when the A in position 3 is exchanged for any other base-pair. Thus, the conditions for detecting specificity changes involving base-pair 3 and amino acid 9 were optimally set. We did not use wild-type residues in positions 1 and 2 of the recognition helix because the operator variant 332, 5’-AATTGTGCGC: might have a tendency to adopt the Z conformation on supercoiled plasmid DNA in viva and thereby impair binding. The results,obtained with the His1 Gln2 Ala5 X9 and ValI Ala2 Ser5 X9 libraries show that at least in these tw.o cases the specific recognition of the A. T pair in position 3 cannot be changed by altering residue 9 of the recognition helix. Our results are thus consistent with the concept that Asn9 enhances the binding of Lac repressor to lac operator by an interaction with a backbone phosphate, possibly between base-pairs 2 and 3 in a manner similar to 1 and 434 repressors (Jordan & et al., 1988; Kisters-Woike Pabo: 1988; Aggarwal et al., unpublished results). Phosphate alkylation protection experiments with Lac repressor and lac operator (Gilbert & Maxam, cited by Barkley & Bourgeois, 1978) have indicated that the phosphates between base-pairs 1 and 2 and between base-pairs 2 and 3 on the 3’ strand of the left half of the wild-type operator are protected against attack by ethylnitrosourea. Ethylation interference experiments (Siebenlist & Gilbert, 1980) with wild-type Lac repressor and suitable mutants may provide further evidence for the function of Asn9 in operator binding. We thank Daniela Tils and Karin Ot’to for excellent technical help and Bob Jack for critically reading this manuscript. This work was supported by Deutsche Schwerpunkt Forschungsgemeinschaft through Proteindesign.

References Aggarwal,

A. K., Rodgers, D. W., Drottar.

M. & Harrison.

M., Ptashne,

S. C. (1988). Science, 242, 899-907.

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M. & Harrison, S. C. (1987). Nature (London) 326, 846-852. Anfinsen, C. B. & Scheraga, H. A. (1975). Advan. Protein Chem. 29, 205-300. Barkley, M. D. & Bourgeois, S. (1978). In The Operon (Miller, J. H. & Reznikoff, W. S.; eds), pp. 1777220, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY. Boelens, R., Lamerichs, R. M. J. N.; Rullmann, J. A. C., van Boom, J. H. & Kaptein, R. (1988). Protein Seq. Data Anal. 1, 487-498. Ebright, R. H.; Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984). Proc. Nat. Acad. Sci., U.S. A. 81, 7274-7278. Hanes, S. D. & Brent, R. (1989). Cell: 57, 127551283. Jordan, S. R. & Pabo, C. 0. (1988). Science, 242, 893-899. Lamerichs, R. M. J. N., Boelens, R.; van der Marel, G. A., van Boom, J. H., Kaptein, R., Buck, F., Fera, B. & Riiterjans, H. (1989). Biochemistry, 28, 2985-2991. Lehming, N., Sartorius, J., Niemoller, M., Genenger, G., von Wilcken-Bergmann, B. & Miiller-Hill, B. (1987). EMBO J.6, 3145-3153. Lehming, N., Sartorius, J., Oehler, S., von WilckenBergmann, B. & Miiller-Hill, B. (1988). Proc. Nat. Acad. Sci., U.S.A. 85: 7947-7951. Lehming, N., Sartorius, J., Kisters-Woike, B.; von Wilcken-Bergmann, B. 8: Miiller-Hill, B. (1990). EMBO J. 9, 615-621. Matthews, B. W., Ohlendorf, D. H., Anderson, W. F. & Takeda, Y. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 1428-1432. Miller, J. H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mondragon; A., Wolberger, C. & Harrison. S. C. (1989a). J. Mol. Biol. 205, 179-188. Mondragon, A., Subbiah, S., Almo, S. C., Drottar, M. & Harrison, S. C. (1989b). J. Mol. Biol. 205, 189-200. B. Oehler, S., Eismann, E. R., KrB;mer, H. & Miiller-Hill, (1990). EMBO J. 9, 973-979. Otting, G., Qian, Y. Q., Billeter, M.; Miiller, M., Affolter, M., Gehring, W. J. & Wiithrich, K. (1990). EMBO J. 9, 3085-3092. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc. Nut. Acad. Sci., U.S.A. 74, 5463-5467. Sartorius, J.; Lehming, N., Kisters, B., von WilekenBergmann, B. & Miiller-Hill, B. (1989). EMBO J. 8, 1265-1270. Siebenlist, U. & Gilbert, W. (1980). Proc. Nut. Acad. Sci., U.S.A. 77, 122-126. Treisman, J., Gbczy, P., Vashistha, M., Harris, E. & Desplan, C. (1989). Cell, 59, 553-562. Wharton R. P. & Ptashne, M. (1987). Nature (London), 326, 888-891. Wolberger, C., Dong, Y., Ptashne, M. & Harrison, S. C. (1988). Nature (London), 335, 789-795.

W. Matthews