Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein

?I. Mol. Biol. (1987) 194, 105-117

Localization of a DNA-binding Determinant in the Bacteriophage P22 Erf Protein Kenan C. Murphy, Linda Casey, Nicholas Yannoutsos Anthony R. Poteete Department of Molecular Genetics and Microbiology University of Massachusetts Medical School Worcester, MA 01605, U.S.,4.

and Roger W. Hendrix Department of Biological Sciences University of Pittsburgh Pittsburgh, PA 15260, U.S.A. (Received 24 October 1985, and in revised form

12 September 1986)

Four amber fragments of the recombination-promoting P22 Erf protein were characterized. The intact Erf monomer contains 204 amino acids. The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in viva. The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf. It breaks down to a stable remnant that is slightly larger than the 149 residue fragment. The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf. Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0. The 95 residue fragment is unstable in vizlo. These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNAbinding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo.

1. Introduction

such as promotion of replication or recombination, is less clear, largely due to a lack of structures solved by X-ray crystallography. The Erf protein of bacteriophage P22 is a member of this class of proteins; it is unusually accessible to structural studies at the lower resolution of electron microscopy due to its distinctive tertiary and quaternary structure. Erf protein binds specifically to single-stranded DNA and promotes homologous recombination of replicating P22 or 2 DNA (Poteete & Fenton, 1983, 1984, and unpublished results). Electron micrographs of Erf reveal a ring-like quaternary structure, consisting of ten to 14 monomeric units (of 204 amino acid residues), with radial projections extending from the outer surface of the ring (Poteete et al.. 1983). An elastase-generated frag-

Many of the key proteins involved in such processes as DNA replication, recombination, and gene regulation exercise their roles in large part through the ways in which they bind to DNA. An appreciation of the structural determinants of DNA binding in such proteins is thus central to our understanding of these processes. Along these lines, a picture of how certain site-specific DNA binding regulatory proteins (2 cT repressor, 1 Cro repressor, and the Escherichia coli CAP transcriptional activator protein in particular) bind to DNA has begun to emerge from structural and genetic studies (for a review, see Pabo & Sauer, 1984). The picture in the case of proteins that are involved in conceptually more complex interactions with DNA, ow%-283(i/87/0501

OF, I3 $03.00/0

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1987 Academic

Press Inc.

(London)

Ltd.

106

K. C. Murphy

of Erf residues I to 136 ment consisting (approximately) was found to retain both the single-stranded DNA (ssDNA)t binding specificity and the ring-like structure of intact Erf, although the radial projections were absent. From these and other observations, it’ was concluded that Erf contains two structurally distinct domains: an amino-terminal portion responsible for DNA binding and maintenance of an oligomeric structure, and a protruding carboxy-terminal portion of unknown function. The existence of a stable amino-terminal fragment’ of Erf with distinctive properties prompted an examination of other amino-terminal fragments, with the goal of answering several questions raised (1) What is the by t,he previous observations. location of the DEA-binding site(s) within the amino-terminal two-thirds of the Erf monomer? (2) What are the boundaries of the two domains? (3) What is the function of the carboxy-terminal domain; in particular, could it be required for the assembly. but not the maintenance, of an oligomerica structure? The additional amino-terminal Erf fragments that are the objects of the studies reported here were generated by amber mutant alleles of the erf gene in a non-suppressing host. An advantage of t,his approach in protein struct’urefunction studies is that, amber mutat’ions can occur in any codon, generating amino-terminal fragments t,ranslation whose size (at least, as primary products) is independent of the occurrence of protease-sensitive sites in the protein. As discussed below, studies of four such amber fragments have provided some answers to the enumerated questions.

2. Materials and Methods (a) Bacteria Eschcrichia

et nl. similarly treated pBR322 (Bolivar rf al.. 1977). Cells wer(b transformed and spread on plates caontaining ampicillin and previously spread with IO’ plaque-forming units of 1 immP22 ~2-5. This procedure selects for hybrid plasmids bearing an EcoRI and PauJI-ended fragment of the P22 chromosome encoding the c2 repressor and Erf (Poteete. 1982). Plasmids found to have the expec&d structures were purified and used as sources of I)NA fragments bearing mutant erf genes. Plasmids t~hat c-an be induced to dire& the synthesis of high levels of Erf ambet were constructed from 3 purified I)Ku‘A fragments fragment,s: (I) t.he Hpnl-EcoRT fragment of P22 I)SA that bears t.he erf gene: (2) t.he replication oripincontaining PstJ-EcoRI fragment from pBR322: and (3) a PstT-PvuIT fragment t,hat contains thtl t.a.r promoter (P,,,) from ptacl2 (Amann rt 01.. 1983). The result,ing plasmids have the same structure as t,he Erf-producing plasmid described by Poteetr & Fenton (1983). (c) beagents,

rrrsdia and t&w+

DEAE-cellulose (Wlex-I)) and Bio-(:rl .a-0.5 m were purchased from Rio-Rad. Hydroxylapat~itr was prepared as described by Muench (1971). LB broth contains. per lit,er. IO g of trypt.one. 5 g of yeast ext,ract. 5 p of NaCl and 1 ml of 1 M-&OH. LB broth is supplemented with 15 p(-r tetracycline/ml for gr0wt.h of the Erf amber fragmrntproducing plasmid-bearing cells. Minimal medium (MM) is M9CAA (Smith h Levine. 1964) with the (tasein hydrosylate omitted, supplemented with I,-histidine and I,-leucine (20 pg/ml each) and 1 pg biotin/ml. TN buffer is 50 mM-Tris HCI 1 mM-EI)TA. (pH 7.4). I nm-2mercaptoethanol, 57, (v/v) glycerol. with various concentrations of T\;aCl. 1% buffer is IO mm-potassium phosphate (pH 6.8). 1 rniM-EDT,k. 1 rn;n-2-mercaptoethanol. SO/, (v/v) glycerol. with various concentra,tions of NaCl. Resuspension buffer is 50 mM-Tris. HCI (pH 7.4). 100 mM-NaCl, 1 m>f-EDTA, 3 miw-2-mercaptoethanol and 10% (I+) glycerol. Storage buffer is Ph’ without I\iaCI 1.4 mM-dithiot.hreit,ol and with substituted for %-mercaptoethanol.

and phage

(d) Phage C~ORS~S

coli W31 IO ZacI“ L8 (Brent & Ptashne,

1981) was used as host in the construction, propagation, and induction of the Erf amber plasmids. Salmonella typhimurium LT2 derivatives MS1363 (ZeuAam414SURE) and TP134 (leuAam414 r- m+ recA- sup’) were used as the permissive and non-permissive hosts, respectively, for phages containing erf-am alleles. P22 strains bearing the amber alleles 12B (Lew & Casjens, 1975), H1046, H1089, H1143, Hl173, HI287 and HI341 (Poteete & King, 1977) were used in these studies. The erf-aml2B allele was in a wild-type background; the others were cl -7 h21. (b) Plasmids General methods for construction, transformation and purification of plasmids were as described (Poteete & Roberts, 1981: Poteete. 1982). DKA fragments were purified as described by Hansen (1981). Phage DXA bearing erf-am alleles was digested with EcoRT and PvuII, extracted with phenol and precipitated with ethanol. It was redissolved in buffer and ligated with t Abbreviations used: ssDh’A, single-stranded DNA; IPTG. isopropylthiogalactopyranoside; SDS/PAGE, SDS/polyacrylamide gel electrophoresis; PET. polyethylenelmine.

MS1363 was grown to a density of 2 x lO*/ml. with aeration in broth a,t 37°C. Phage were added at, a multiplicity of 5 of each parent (IO for infections with single phage strains). Aeration was continued for 2 h. b> which time complete or nearly complete lysis was generally observed in crosses with ~1~ phage. Lysatrs were shaken with chloroform and tit,ered on permissive and non-permissive cells. Crosses that failed t.o produce a proportion of wild-type progeny significantly above the background of reversion of the parents (typically about 0*00020&) were taken as evidence t’hat the 2 mutations involved occupy the same site. Results of the crosses sorted the 7 amber mutations into 4 sites: (1) H1046. Hl143. and H1341: (2) Hl173; (3) HlO89: (4) 12B and HI287 (data not shown). (e) DNA

sequencing

of Erf amber mutations

DKA sequence determinations were by the method of Maxam & Gilbert (1980). The amber fragment-producing plasmids described above served as sources of DNA fragments for sequencing the amH1046 and amH1287 mut,at.ions. In the case of amH1046, the DKA sequence encoding Erf from the amino terminus t,o codon 110 (YW Fig. 1) was the same as wild type, except for a G/C t,o A/T

DNA-binding

Determinant in P22 Erf Protein

transition that changes codon 96 from CAG to TAG. In the case of amH1287, the DNA sequence encoding Erf from codon 125 to the carboxy terminus was the same as wild type except for a G/C to A/T transition that changes codon 191 from CAG to TAG. In these cases, DNA sequences were determined for 1 strand only (data not shown). (f) Purification

of Erf amber fragments

Plasmids producing Erf amber fragments under control of p,*c were placed in sup’ cells containing the lacIq mutation, which overproduces the Zac repressor (MiillerHill et al.. 1968). In such an environment, P,,, is repressed and the fragments are not produced at high levels. With the Erf amber fragments under repressor control, host cells can be grown to a high density before the fragments are induced with isopropylthiogalactopyranoside (IPTG). Such a scheme is necessary, as high concentrations of Erf, and possibly of the fragments, are detrimental to the cells. Cells bearing plasmids that produce Erf or Erf amber fragments were grown by swirling at 37°C in 500 ml of LB broth with 15 to 20 pg tetracycline/ml in a 2 1 flask. When the culture reached a density of about 2 x 10’ cells/ml, I ml of 0.1 M-IPTG was added and swirling was continued for 25 h. The cells were collected by centrifugation, resuspended in 3 to 5 ml of resuspension buffer and either frozen at - 70°C for later use or Iysed immediately. In the ensuing procedures, the extent of purification at each step was monitored by SDS/polyacrylamide gel electrophoresis (SDS/PAGE), which served as an assay for Erf and the amber fragments. (i) Method I The purification of F-149 was as described for Erf (Poteete & Fenton, 1983), with the exception that F-149 was divided equally between the polyetheleneimine (PEI) supernatant and precipitate. The protein was isolated from both fractions; no differences in the properties of the 2 isolates were observed. The yield of F-149 was about 1.5 mg from 6.3 g of packed cells. In the case of F-130, a cell extract was prepared in a manner identical to bhat described for Erf and F-149. PET (20.4 ml of a 10% (w/v) solution) was added to 320 ml of cell extract, the solution was stirred overnight and centrifuged for 20 min at 8000 revs/min in a GSA rotor. SDS/PAGE revealed that F-130 was predominantly in the PEI supernatant, though a significant amount had been precipitated with PEI. The protein was extracted from the PEI precipitate with TN500 buffer (TN made 500 mM-NaCl), precipitated with ammonium sulfate and resuspended in 6 ml of TN500 buffer (fraction A). To the PET supernatant, ammonium sulfate (0.37 g/ml) was added, the protein was precipitated and suspended in TN25 buffer and dialyzed against the same buffer overnight. A precipitate developed in the dialysis bag and was collected and redissolved in 42 ml of TN500 buffer. The dialyzed supernatant was discarded. This fra,ction was combined with fraction ,4 and roncentrat,ed to 1 ml by precipitation with ammonium sulfate. One-half of the preparation was then applied to a Rio-Gel A-O.5 m column (1.5 cm x 45 cm) using TN500 as the elution buffer. F-130, which appeared in the void volume. was collected and dialyzed against PN25 buffer (2 h to minimize precipitation) and applied to a phosphocellulose column (1.5 cm x 23 cm) equilibrated with PN25 buffer. F-130 flowed through the column and was collected in the wash (33 ml). The protein solution

107

was concentrated to 15 ml using an Amicon single-use filtration unit, dialyzed against TN25 buffer and applied to a DEAE-cellulose column (1 cm x 17.5 cm). The column was washed extensively with TN25 buffer and an 80 ml gradient of 25 to 400 mm-NaCl in TN buffer was applied. F-130 eluted between 180 and 200 mM-NaCl and was dialyzed against 0.01 M-sodium phosphate (pH 7.2). The other half of the preparation was then purified using the procedures just described. The 2 halves of the preparation were then combined and applied to a hydroxylapatite column (1 cm x 17.5 cm) equilibrated with 0.01 m-sodium phosphate (pH 7.2). A 200 ml gradient of 0.01 to 0.3 M-sodium phosphate (pH 7.2) was applied and 3 peaks were obtained. None of the peaks. however, contained F-130 as detected on SDS/PAGE and a second gradient of 200 to 500 mm-potassium phosphate (pH 7.21, was applied. Neither this nor a third gradient of 500 to 750 mw-potassium phosphate (pH 7.2) eluted F-130. The column was washed with successive 2-columnvol. washes of 1 M-, 2 M- and 3 iw-NaCl in lOOrn~potassium phosphate (pH 7.2). F-130 was eluted with 1 M-sodium citrate (pH 7.2), and dialyzed extensively against storage buffer overnight. The prot’ein was precipitated with ammonium sulfate (95oj), stirred overnight at 4 “C, redissolved in 2 ml of storage buffer and dialyzed against the same buffer. The yield was about 1 mg from 7 g of packed cells. Portions were stored at -70°C.

(ii) Method 2 Cells from 2 1 of culture containing Erf, F-149 or F-130 were collected by centrifugation and resuspended in 20 ml of resuspension buffer. An equal vol. of 1 mg hen eggwhite lysozyme/ml in 0.125 M-Tris. HCl (pH 7.5), 8.3% (w/v) sucrose was added. The solution was stirred in the refrigerator; all subsequent steps were carried out at 4°C. After 5 min, 0.8 ml of 0.5 M-EDTA was added. followed 25 min later by 45 ml of 1% (w/v) Brij-58, 50 DIMTris . HCl (pH 7.5) 2 mm-dithiothreitol. After an additional 30 min of stirring, the extract was centrifuged in a SW-70 rotor at 40.000 revs/min for 2 h. The supernatant was brought to 25% saturation with ammonium sulfate, stirred for 0.5 t,o 1 h and centrifuged in a SW-70 rotor at 25,000 revs/min for 1 h. The supernatant was collected and brought to 50% saturation with ammonium sulfate, stirred for 1 to 2 h and centrifuged as before. The precipitate was resuspended in 10 to 15 ml PN buffer and placed direct.ly on a hydroxylapatite column (15 cm x 1.5 cm) that had been equilibrated with PN buffer. The column was washed with PN buffer until the absorbance of the eluent fell below 0.05. This was followed by a wash with phosphate (pH 7.2), 2 mw-dithio500 IUM-pOtaSSiUUI threitol, 5% glycerol. After the absorbance fell below 0.05 (approx. 2 column vol.), a 3rd wash of 1 ivr-NaCl in PN buffer was applied to the column. Erf, F-149 and F-130 were eluted from the hydroxylapatite column with 250 mmsodium citrate (pH 7.2), 2 m#-dithiothreitol. 5oj, glycerol. The peak fractions were combined (20 to 25 ml) and dialyzed against TN buffer for 4 to 8 h with 3 changes of buffer. Cold water (1 to 2 vol.) was t,hen added to the dialysate to bring the conductivity down to that of TN buffer. The protein solution was then applied to a 5 ml DEAE-cellulose column equilibrated with TN buffer. Erf, F-149 and F-130 eluted near the middle of a 25 to 306 rnx-NaCl 50 ml gradient in TN buffer. The peak was collected and concentrated with a Centriconmicroconcentrator (Amicon) to 1 to 2 ml. The sample was then

108

K. C. Murphy

applied to a Bio-gel AO.5 m column (60 cm x 1.5 cm) in TN buffer. Erf, F-149 and F-130 appeared in the void volume, were collected and concentrated as before. Yields were typically 10 to 15 mg of protein. Portions were stored at - 70°C.

(g) Carboxy-terminal sequencing of Erf and the amber fragments Carboxypeptidase Y (Boehringer) was dissolved in 1 ml of 0.01 M-sodium phosphate (pH 7.2), and dialyzed against the same buffer. Erf, F-149 and F-130 were dialyzed against 0.05 M-N-ethylmorpholine (pH 5.5). The reactions contained 1 nmol of Erf (or amber fragments) and carboxypeptidase Y with a substrate/enzyme molar ratio of 80 : 1. Reactions were carried out at 37°C in 0.05 M-N-ethylmorpholine (pH 5.5), in a total volume of 0.20 ml. At 10, 30 and 60 min, 0.05 ml portions were removed and the reaction was stopped by boiling for 5 min followed by freezing in solid CO, and lyophilization. Other portions (10 ~1) were also removed and analyzed on SDS/polyacrylamide gels to examine the extent, if any, of contaminating protease activity. Amino acid analysis of the lyophilized samples was done at the University of Massachusetts Medical Center Protein Chemistry Facility. The amounts (in picomol) of each amino acid released at 10, 30 and 60 min incubation with carboxypeptidase Y are listed in Table 1. A few random time points were contaminated with serine and glycine as judged by their absence in latter time points.

(h) DNA-binding

assays

Detection of Erf fragment,-DNA complexes by agarose gel electrophoresis and assays involving protection of DNA from S, nuclease were carried out as described (Poteete & Fenton, 1983). DNA-protein complexes were detected using nit,rocellulose filters (Millipore, HAWPO025). Untreated filters were soaked in PE buffer (10 mM-potassium phosphate (pH 6.0), 2 mM-EDTA, 5 mM-NaCl, 0.5 mM-dithiothreitol) for at, least 30 min prior to use. Filters were washed 3 times with 2 ml of PE buffer prior to filtration of the reaction mixtures. DNA substrate was prepared by endlabeling a 95 base-pair EcoRI fragment containing the P,,, promoter and isolated on a bis-acrylylcystamine polyacrylamide gel (Hansen, 1981) An end-labeled linear pBR322 backbone (with the same specific activity of the P,,, fragment) was used to establish the DNA concent.ration by comparison with known standards run on an agarose gel and stained with ethidium bromide. DNA substrate was boiled for 5 min and immediately placed on ice for 10 min prior to addition of protein. Reaction mixtures (500 ~1) contained 46 PM-ssDNA with various amounts of Erf or Erf amber fragments in PE buffer. After 30 min at room temperature, the reaction mixtures were filtered through the nitrocellulose filters and washed 4 times wit,h 2 ml of PE buffer. In the absence of protein, more than 97% of the counts washed through the filter. The counts transferred to t,he filters were corrected for absorbtion of DNA by the reaction tubes or pipette tips.

(i) Electron microscopy Negative staining and electron microscopy of Erf fragments were carried out as described (Poteete et al.. 1983).

et al. (j) [“5S]methionine

labeling of proteins in vivo

Cells containing the amber fragment-producing plasmids were grown to approximately 2 x 10s cells/ml with aeration in MM. IPTG was added to a final concentration of 1 mM. After incubation at 37°C for 10 min, 5 ~1 of [35S]methionine (10 PM. 1139 Ci/mmol) was added t,o 50 ~1 of each culture. Following a 5 min incubation at 37”C, 5 ~1 of the mixture was removed and mixed with an equal volume of 2 xSDS/sample buffer (Laemmli. 1970) and immediately heated to 90°C for 2 min. To the remainder, 5 ~1 of unlabeled methionine (25Opg/ml) was added and incubation was continued at 37°C with aeration. Samples (5~1) were removed at 15 and 60 min and mixed with equal vol. of 2 x SDS/sample buffer. The samples were heated to 90°C for 2 min. Portions (2 ~1) of each sample were placed on a gradient gel (10% to 22 “0; SDS/polyacrylamide acryllbisacryl, 30 : 0.8) and subjected to electrophoresis (6 V/cm) for 20 h. The gel was fixed in 200/, ethanol/70/;, acetic acid (v/v) and washed in the same solution made lOTo glycerol. The gel was dried and exposed at -70°C for 4 days.

3. Results (a) Erf amber jkzgment production and puri$cation

In order to obtain a series of amino-terminal fragments of Erf. we placed amber mutant alleles of the erf gene in plasmids under the control of a strong inducible promot,er. First, a collection of seven independently isolated amber mutations in the P22 erf gene was sorted by t,wo-factor crosses into four different sites. DNA fragments containing the erf genes of phage mutants representing the four sites were then used in plasmid constructions and two of them were sequenced as detailed in Materials and Methods. The other two had been sequenced already (Poteete, 1982). The sequence of the wildtype erf gene, and the alterations borne by the four amber alleles are shown in Figure 1. All four mutations

involve

a transition

from

a glutamine

codon (CAG) t,o the amber codon (TAG). The codons affected are at positions 96 (amH1046), 131 (amH1173), 150 (amH1089) and 191 (amH1287). The amber fragments produced by these genes should contain 95, 130, 149 and 190 residues, respectively; wild-type Erf has 204 residues. We assume that the amino-terminal methionine residue indicated by the DNA sequence is removed from the amber fragments as it is from wild-type Erf (Poteet’e et al., 1983). For convenience.

we designate

the fragments as F-95, F-130, F-149 and F-190. Following induction, cells bearing the amber mutant plasmids produce large a,mounts of the amber fragments. The apparent sizes of the fragments are qualitatively consistent with the positions of the amber mutations in the erf DNA sequence, as shown in Figure 2, which is a photograph of a stained SDS/polyacrylamide gel run with samples induced cells in

plasmid-bearing

prepared

,bT

heating

intact,,

SDS-contammg buffer. Noncells, and tjhr amino-terminal

elastase fragment of Erf are included as cont,rols. It’ can be seen that’ the elastase fragment is inter-

DNA-binding

Determinant

in P22 Erf Protein

109

10 Ser Lys Glu Phe Tyr Ala Arg Leu Ala Glu Ile Gln Glu His Leu Asn Ala Pro Lys ATG AGC AAA GAG TTT TAC GCA AGA CTT GCT GAA ATT CAG GAG CAT CTG AAT GCG CCA AAG

30 20 Asn Gln Tyr Asn Ser Phe Gly Lys Tyr Lys Tyr Arg Ser Cys Glu Asp Ile Leu Glu Gly AAT CAG TAC AAC TCG TTT GTT AAA TAC AAA TAC CGC AGT TGT GAG GAC ATT CTT GAG GGT 50 40 Val Lys Pro Leu Leu Lys Gly Leu Phe Leu Ser Ile Ser Asp Glu Ile Val Leu Ile Gly GTT AAG CCA CTA CTG AAA GGT CTG TTC CTG TCT ATC AGT GAT GAA ATC GTG CTG ATT GGC

60

70

Asp Arg Tyr Tyr Val Lys Ala Thr Ala Thr Ile Thr Asp Gly Glu Asn Ser His Ser Ala GAC CGT TAT TAC GTC AAG GCC ACA GCG ACC ATT ACA GAT GGT GAA AAT AGC CAT TCA GCA 90 80 Ser Ala Ile Ala Arg Glu Glu Glu Asn Lys Lys Gly Met Asp Ala Ala Gln Val Thr Gly AGC GCT ATA GCG CGA GAA GAA GAA AAC AAG AAG GGA ATG GAT GCA GCT CAG GTA ACG GGC (ailO46) 100 110 Ala Thr Ser Ser Tyr Ala Arg Lys Tyr Cys Leu Asn Gly Leu Phe Gly Ile Asp Asp Ala GCT ACA AGC TCT TAC GCT CGC AAA TAT TGC CTT AAC GGT TTG TTT GGT ATC GAC GAC GCC 120 130 Lys Asp Ala Asp Thr Glu Glu His Lys Gln Gln Gln Asn Ala Ala Pro Ala Lys Gln Thr AAA GAC GCT GAT ACT GAG GAG CAC AAA CAG CAG CAG AAT GCA GCA CCT GCT AAG CAA ACT (arrH:173) 140 150 Lys Ser Ser Pro Ser Ser Pro Ala Pro Glu Gln Val Leu Lys Ala Phe Ser Glu Tyr Ala AAA TCA TCG CCT TCC TCC CCT GCT CCT GAA CAG GTT CTT AAG GCA TTC AGT GAA TAT GCA (ad+:0891 160 170 Ala Thr Glu Thr Asp Lys Lys Lys Leu Ile Glu Arg Tyr Gln His Asp Trp Gln Leu Leu GCA ACA GAA ACG GAC AAG AAA AAG CTA ATT GAG AGA TAC CAG CAC GAC TGG CAA TTA TTG 18) 190 Thr Gly His Asp Asp Glu Gln Thr Lys Cys Val Gln Val Met Asn Ile Arg Ile Asn Glu ACT GGT CAC GAT G4T GAG CAG ACA AAA TGC GTT CAG GTA ATG AAT ATC AGA ATA AAT GAG T

(antii287)

200 Leu Lys Gln Val Ala CTT AAA CAG GTG GCT TAA TGA

Figure 1. The sequence of the erf gene is shown together with the predicted amino acid sequence of the Et-f protein. The codon numbering st)arts with the AGC (serine). The positions of the 4 amber mutations at codons 96, 131, 150 and 191 are indicated.

mediate in mobility between F-130 and F-149, consistent with its having residues 1 to 136, as has been estimated (Poteete et al., 1983). The F-95 fragment is not seen clearly in the photograph; it is produced, but is unstable in vivo (see below). The largest Erf amber fragment, F-190, is unstable in vitro. In cell lysates prepared by sonication or by lysozyme and non-ionic detergent treatment, it quickly breaks down to a size slightly larger than that of the F-149 fragment, This breakdown product behaves in the same way as intact Erf and F-149 on gel filtration columns, indicating an oligomeric structure for F-190. Our preparations of intact Erf, subjected to the same initial purification steps, always contain a small amount of an amino-terminal fragment of the same size as the breakdown product of F-190 (fragment A, Poteete et al., 1983). Evidently, the absence of the carboxy-terminal 14 amino acid residues greatly

enhances the susceptibility to proteolysis of some or all of the residues that normally constitute the carboxy-terminal domain of Erf, back to the vicinity of residue 150. Different methods were employed for the initial purifications of F-149 and F-130. F-l 49 was purified by methods previously employed for Erf, including gel filtration and chromatography on phosphocellulose and DEAE-cellulose. F-130, unlike F-149 and Erf, does not bind to phosphocellulose at pH 6.8 in low salt, so a different purification protocol was employed. Both fragments are apparently stable in vitro, and were purified to greater than 95% apparent homogeneity as detailed in Materials and Methods. Binding to phosphocellulose by Erf and F-149 and the lack of binding by F-130 suggest that a region within residues 131 to 149 is important for Erf s interaction with negatively charged groups.

110

K. C. Murphy

et al.

Figure 2. Production of the Erf amber fragments from plasmids. Cultures of the plasmid-bearing cells were grown and buffer. subjected to induced with IPTG. Following concentration, portions were heated in SDS/sample SDS/polyacrylamide

gel electrophoresis

(12.5%; acryl/bisacryl

Indeed, as shown below, F-130 has a significantly lower affinity than does Erf or F-149 for ssDNA. In order to eliminate the possibility that differences in DNA binding between the two fragments might be due to differences in treatment during purification, a single purification scheme was devised for both fragments and Erf. It was found that intact Erf, F-149 and F-130 bind tightly to hydroxylapatite, presumably through binding to the positively charged Ca’ + ions, since Erf and both fragments were retained after treatment with buffers of high NaCl concentration and eluted with a chelating agent, citrate (Bernardi, 1971). The specificity of this tight binding resides in the amino-terminal domain of Erf (residues 1 to 130), since this is the only region common to all three proteins. After hydroxylapatite chromatography, Erf and the fragments showed single bands on SDS/polyacrylamide gels. A gel containing samples of ammonium sulfate and hydroxylapatite fractions of both F-149 and F-130 is shown in Figure 3. The other steps in the procedure involve gel filtration and chromatography on DEAF,-cellulose, as detailed in Materials and Methods. Isolation of the smallest Erf fragment, F-95, was

(20 : 1)) and stained with Coomassie blue.

not possible due to its instability in vim (see below). The fragment could be detected in SDS/polyaerylamide gel samples made by heating whole induced cells in SDS/sample buffer (see Fig. 4). However, cell extracts prepared by the methods used for the other amber fragments showed nearly total loss of F-95 on SDS/polyacrylamide gels. This would seem to indicate that some residues between 96 and 130 (inclusive) are necessary for a stable amino-terminal domain. Attempts to isolate F-95 in the presence of the protease inhibitor phenylmethylsulfonylfluoride and/or the use of alternate extraction procedures (sonicated lysates) were unsuccessful. (b) &ability of the amber fragments in vivo In order to examine the stabilities of the Erf amber fragments in aivo, a pulse-chase labeling experiment was carried out]. Cells bearing amber fragment-producing plasmids were induced with IPTG, and pulse labeled with [35S]methionine for five minutes. Excess non-radioactive methionine was added and incubation was continued for 15 and 60-minute chase periods. SDS/PAGE of proteins extracted from the cells, followed by autoradiography gave the results shown in Figure 4. F-190,

I

3

2

4

5

Figure 3. Purification of F-149 and F- 130: lane 1. ammonium sulfate fraction of F-149 extract: lane 2. F-149 after elution from hydroxylapatitr: lane 3. ammonium sulfate fraction of F-130 ext,ract; lane 4. F-130 aft’er elution from hydroxylapat’ite: lane 5, molecular weight standards: ovalbumin (43 1~). chymotrypsinogen (25.7 K). lactoglobulin (18.4 K). lysozyme (14.3 K) and bovine trypsin inhibitor (6.2 K). Percentage of total sample put on gel: ammonium sulfate fractions. 0.005 96: hydroxylapatite fractions, 0.0025 “/o. K. lo3 units of molecular weight.

ErfamH1287amHl089amHlI73amHlO46-

I

2345678

Figure 4. Pulse-chase [35S]methionine

9

IO II

I2

I3

I4

labeling experiment following Erf and Erf amber fragment induction. Pulse period was 5 min. Lane 1, Erf control (no induction); lane 2, Erf; lane 3, F-190; lane 4, F-190, 15 min chase; lane 5, F-190. 60 min chase; lane 6, F-149; lane 7, F-149, 15 min chase; lane 8, F-149, 60 min chase; lane 9. F-130; lane 10, F-130. 15 min chase; lane 11, F-130. 60 min chase; lane 12. F-95; lane 13, F-95, 15 min chase; lane 14, F-95, 60 min chase. The lower molecular weight bands shown in the induced lanes are products of genes other than erf. that are present on t,hr plasmids.

112

K. C. Murphy et al.

Table 1 Amino acids (picomol) released from Erf and the amber fragments after treatment with carboxypeptidase Y Residue

1ot

Asp Glu Ser/Gln Gl$ His Arg Thr Ala Pro Tj? Val Met ($3 Ik Leu Phe LgS

12.5 10.5 ‘47.1 “9.2

Erf 30

60

10

: 7.6 :5,1

10.5 14.3

42.0 98.3 17.8

94.3 8.5

36.2 28.3

F-149 30

60

71.3 10.6

1064l 20.7

24.9 62.9

52.1 120.2

6.1

5.0

i 10 15.1 X.9 ;i6.0 33.1 13.0

F-130 30 11.6 11.5 42.0

60

10.7

17.1 (3-S 69.3 53.0 14.3

11.4 11.2

10.1 10.8

11.X 157

6.X ii.5

74 5%

X.8 6%

f2.6

109.7 11.2 6.9 71.3

66.1

104.8 12.1 51 71.3

12.3

id

11.1

6.5

5.5

55

12.8

9.1

I I.1

X.6

6-l

5.3

Trp and Am were not identified. t 10, 30, 60 are incubation times in minutes. 3 A dash indicates less than 5 piromol released

F-149 and F-130 did not break down during the incubation period. Evidently, F-190 is more susceptible to proteolytic digestion after cell disruption. On the other hand, the smallest fragment, F-95 (which did not accumulate to the same extent that the others did in the 10 min incubation with IPTG), is lost during the Iii-minute chase period. (c) Amino acid sequences of Erf, F-149 and F-130 In order to examine the carboxy terminus of Erf and the intermediate amber fragments, the proteins were treated with carboxypeptidase Y. Table 1 lists the amounts of amino acids released from Erf, F-149 and F- 130 at different times following treatment with carboxypeptidase Y. At early times, alanine and valine were released in greatest amounts from intact Erf, as predicted from the DNA sequence. For the F-149 fragment, glutamic acid, alanine and proline were found to be released to the greatest extent after exposure t,o carboxypeptidase Y, as predicted by the amber mutation amH1089 in the erf sequence. In the case of the amH1173 mutation, the predicted carboxy terminus for the amber fragment consists of two A peak representing glutamines. consecutive glutamine (or serine) was found in the highest amount aft’er treatment of F-130 with carboxypeptidase Y. These results, combined with the apparent stability of F-130 and F-149 in Vito and in vitro. suggest strongly that the purified fragments have the carboxy-terminal amino acid sequences predicted by the DNA sequences. Moreover, the identity of the amino-terminal sequences of Erf and two amino-terminal fragments generated by proteolysis (Poteete et aE., 1983) indicates that the amino

terminus of the protein is stable. Hence, there is no reason to t,hink t’hat the amber fragments would differ in amino-terminal sequence. We conclude that F-130 and F-149 probably consist of Erf amino acid residues 1 to 130 and 1 to 149, respectively. (d) Morphology of the Erf amber fragments The purified amber fragments F-149 and F-130 were examined by electron microscopy and found to have the ring-like quaternary structure of intact Erf, as shown in Figure 5(a) and (b). This observation allows us to extend our earlier conclusion that the C-terminal portion is not, required for stability of the ring structure: since the amber peptides have never contained the C-terminal amino acids, the N-terminal regions themselves are sufficient to allow the N-terminal domain to fold and the ring t#o assemble from its subunits. Like the elastase-generated amino-terminal Erf fragment, the amber fragment rings appear to lack the more prominent radial extensions that are present in intact Erf (Fig. 5(c); Poteete et al., 1983). in agreement with the idea that the C-terminal portion of the polypeptide forms these structures. Like the elastase fragment,, both amber fragments occasionally appear in the edge-on view: these are the dumb-bell shaped images that, appear more prominently in the area of thicker negative st,ain (Poteete et al.. 1983). There are also occasiona, intermediate views that’ were interpreted as molecules t,hat are tilted with respect t’o the grid and in some case only partially delineated by the thin negative stain. In contrast to the multiple views shown by the amber fragment molecules, the wildtype Erf protein is always seen in the face-on view

DNA-binding

Determinant in P22 Erf Protein

113

(b) Figure 5. Electron micrographs of Erf and amber fragments of Erf, negatively stained with uranyl formate. (a) F-130; (b) F-149; (c) wild-type Erf. The phage P22 virions seen in (a) and (b) were included to aid in the spreading of the stain.

Magnification * 120.000x

(Fig. 5(c)). intact and surface of they differ ring.

This difference in the ways in which the truncated proteins can attach to the the grid reinforces the contention that from each other at the periphery of the

(e) Gel assay of Erf amber fragment-DNA complex formation A complex of Erf bound to heat-denatured, linearized pBR322 DNA has a lower electrophoretic mobility in agarose gels than does uncomplexed DNA. This difference in mobility serves as an assay for binding. Incubation of saturating amounts of Erf (4 pg of protein/O.15 pg of DNA) with ssDNA substrate for ten minutes at room temperature generates the high molecular weight complex shown in lane 3 of Figure 6. That this band is due to protein binding is revealed in lane 4 where a sample identical with that shown in lane 3 is incubated an additional ten minutes with proteinase K at 37°C. Incubation without proteinase K at 37°C has no effect on the complex (data not shown). Proteinase K releases ssDPIv’Aindistinguishable from untreated ssDNA. Lane 5 reveals that Erf has no effect on the mobility of linear double-stranded DNA. This is in agreement with previous observations (Poteete & Fenton, 1983). Erf amber fragments F-149 and F-130 were tested in the same way. Lane 6 of Figure 6 shows complex formation after incubation

of F-149 with ssDNA for ten minutes at room temperature. The greater mobility of the F-149ssDNA complex with respect to the Erf-ssDNA complex reflects either the binding of a lower molecular weight protein or a substantial difference between the conformations of ssDNA induced by the fragment and by intact Erf. The stoichiometry in both cases is similar; that is, complex formation is complete at a ratio of protein/DNA of about 20 : 1 (w/w). As with intact Erf, lower amounts of F-149 result in a population of diffuse bands intermediate between free and totally complexed ssDNA. Such behavior represents non-co-operative binding of the proteins to ssDNA. Treatment of F-149-ssDNA complex with proteinase K released ssDNA (Fig. 6, lane 7). As was the case with intact Erf. F-149 has no effect on linear double-stranded DNA (Fig. 6, lane 8). Incubation of F-130 with ssDNA at the same protein/DNA ratio (20: 1) does not produce a discrete complex as was seen for Erf and F-149. At this concentration, F-130 causes the ssDNA band to become slightly diffuse, suggesting some interaction between F-130 and ssDNA (Fig. 6, lane 9). Incubation with greater amounts of F-130 (protein/DNA z 75 : 1) led to a smear on the gel indicative of a series of diffuse bands (data not shown). Ultimately, at a protein/DNA ratio of 300 : 1, F-130 led to formation of a complex that was too large to enter the gel, a property not exhibited

114

K. C’. Murphy

1

2

3

4

5

6

et al.

7

8

9

10 11

Figure 6. Binding of Erf and Erf amber fragments to single-stranded DEA. Substrat,e is pBR322 digested with C’laI (approx. 0.15 pg/lane). Protein, when present’, was 2 to 3 pg. Lane 1. native DNA; lane 2. denatured DNA: lane 3. denatured DPU’A+Erf; lane 4; denatured DNA+Erf+proteinase K; lane 5. uative DNA+ Erf, lane 6. denatured DEA+ F-149; lane 7, denatured DNA+F-149+proteinase K; lane 8, native DNA + F-149; lane 9, denatured DNA + F-130; lane 10, denatured DEA+ Erf+F-130; lane 11. denatured DNA + F-149+F-130.

by either intact Erf or F-149. These results suggest that F-130 has a lower affinity for ssDNA when compared to Erf or F-149 and, unlike Erf or F-149, causes aggregation of the linear ssDNA molecules at high concentration. Addition of F-130 had no effect on the abilities of intact Erf or F-149 to bind to ssDNA (Fig. 6, lanes 10 and 11). This result indicates there is nothing in the F-130 preparation that inhibits binding. (f) Measurement

of DNA-binding

afinities

The ability of Erf and the amber fragments to bind to nitrocellulose was used to measure their affinities for ssDNA. In order to ensure that the assay measured binding between single Erf or fragment rings and DNA molecules, small DNA fragments were employed at a concentration low relative to the concentration of protein required to obtain measurable binding. The DNA substrate employed in the experiment of Figure 7 was a heatdenatured 95 base-pair fragment. The resulting curves appear to be first-order in their dependence

on protein concentration, consistent with binding of single oligomeric proteins. The concentration of monomer at which half-maximal binding was observed was 1.8 x 10m9 M for Erf, 1.0 x 10m9 M for F-149, and 4.0 x lo-* M for F-130. Thus, Erf and F-149 have nearly the same affinity for ssDNA, while that of F-130 is significantly lower. The slightly greater apparent affinity of F-149 relative to Erf may be due to small errors in the determination of protein concentrations. The small differences in amounts of DNA bound at saturation in experiments with the three proteins presumably reflect the different’ efficiencies with which the complexes are retained on the filters under the conditions tested. One possible interpretation of the relatively large amount of F-130 required to observe half-maximal binding in the experiment of Figure 7 is that the purified F-130 is largely inactive, unlike the F-149. Thus, although the proteins were purified by the same procedures, we could be looking at binding by 2% of the nominal amount of F-130 present in the reaction, and comparing it, to binding by SO%, of

DNA-binding

Determinant in P22 Erf Protein

115

a) I

/

F-149

80

Figure 8. Schematic drawing of the Erf oligomeric ring (assuming 12 subunits); broken line represents ssDNA.

1

I 50

I 100

I 150

[Protein]

(rnht)

I 200

I

Figure 7. Nitrocellulose filter binding assays with Erf, F-149 and F-130. The DNA concentration is 46 PMssDNA. (a) Protein. 0 to 20 nm; (b) protein, 0 to 200 nm. the F-149. The results of S, nuclease protection experiments lead us to think that this is not the case. Under conditions including a DNA concentration sufficient to ensure stoichiometric binding, equimolar amounts of F-130 and F-149 protect equal amounts of DNA from digestion, within the limits of experimental error (data not shown). We conclude that the difference in DNA-binding apparent in Figure 7 is due to F-130 having an affinity lower than that of F-149 for ssDNA.

4. Discussion As a result of examining the properties of aminoterminal fragments of the Erf protein, we are able to assign functional roles to parts of the Erf protein structure. (1) Some residues between 95 and 131, but no residues beyond these, are required for the formation of a stable amino-terminal domain and ring oligomer. (2) Some residues between 130 and 150 are involved in forming or stabilizing a DNAbinding determinant. Previous results (Poteete et al., 1983) suggest in addition that residues beyond 137 are not part of the DNA-binding determinant. (3) Some residues between 191 and 204 are required for the formation of a stable carboxy-terminal domain. (4) The carboxy-terminal domain is not

required for the formation of a stable oligomer. A sketch of the Erf molecule that incorporates these observations is shown in Figure 8. In it, the Erf monomer is implicitly divided into three parts: amino-terminal domain, DNA-binding segment, and carboxy-t,erminal domain. In what follows, we present the reasoning behind these structural assignments. Finally, we compare this picture with what is known about structure-function relationships in some other ssDNA-binding proteins. The carboxy-terminal part of the Erf monomer is designated as a domain, implying that it is a discrete, ordered structural element of the protein. A number of observations support, this view. First, Erf lacking its carboxy-terminal 14 amino acid residues is considerably more labile than is intact Erf. Both of them break down in extracts, at different rates, into amino-terminal fragments of approximately 150 residues. In the case of the breakdown product of intact Erf (fragment A), we determined previously by sequencing that it is an amino-terminal fragment (Poteete et aE.. 1983); we assume that the F-190 breakdown product is also amino terminal. The size of 150 residues is based on its mobility in SDS/PAGE, which is slightly lower than that of F-149. The fact that Erf lacking its last 14 amino acid residues is hypersensitive to proteases at a site 40 residues away in the primary and possibly at undetected sites in structure, between, suggests strongly that at least some of the 14, as well as all other residues back to position 150, are part of an organized structure. A second indication of the structural organization of the carboxy-terminal part of Erf comes from previously reported studies of DNA binding by Erf and the elastase-generated amino-terminal fragment. Both bind DNA; mild heat treatment of intact Erf, but not of the fragment, caused it to form large aggregates with DNA, presumably held together by protein-protein binding (Poteete et al., 1983). This observation suggests that the change on heating involves a structural transition (perhaps denaturation) in a carboxy-terminal domain. A third hint of organization in the carboxy terminus comes from the visualization by electron microscopy of stain-

116

K. C. Murphy et al.

excluding structural elements, projecting from the oligomeric ring of intact Erf, that are missing or less prominent in preparations of three different aminoterminal fragments. The properties of the amber fragment F-190 show that the carboxy-terminal domain of Erf includes some, possibly all, of the last few amino acid residues in the sequence. The location of the aminoboundary terminal of the carboxy-terminal domain’s sequence determinants is more ambiguous. One simple hypothesis would place it, somewhere in the vicinity of residue 150, in effect defining the carboxy-terminal domain as those residues lost at a high rate from F-190 (i.e. unable to form a stable structure in the absence of the last 14 residues). A problem with this definition is that, it is arbitrary. It depends on the specificity of the proteases in an E. coli extract; other proteases, such leave different amino-terminal as elastase, remnants. It is difficult to see how this issue can be settled in the absence of a high-resolution structure. The function of the Erf carboxy-terminal domain remains undetermined. Our previous results showed that the domain, as defined above, is not essential for single-strand-specific DNA binding or for the maintenance of a stable ring oligomer (Poteete et al., 1983). To this we now add two negative findings on the function of the carboxy-terminal domain: removing it has little or no effect on Erfs affinity for single-stranded DNA; and it is not essential for the formation of a ring oligomer. The carboxy terminus of the protein is essential for Erf function in vivo, however, as shown by the fact that the amber mutation that generates F-190 (which is stable in vivo) confers an Erf- phenotype. The peripheral location of the carboxy-terminal domain. and its apparent dispensability in DNA-binding, together suggest that its role is to interact with other components of the phage recombination system. Recent studies (unpublished) have uncovered the existence of an accessory recombination gene that maps to the right of erf, and is thus a candidate for such a component. The amino-terminal domain is the best-defined part of the Erf molecule. It forms a proteaseresistant ring oligomer of ten to 14 monomeric units as estimated by attempts to count the carboxyterminal projections of intact Erf in electron micrographs. The observed stability of F-130 and the extreme instability of F-95 permit a relatively unambiguous placement of the carboxy-terminal boundary of this domain between residues 95 and 130. It remains a possibility that F-95 breaks down into a stable fragment still capable of maintaining an oligomeric structure, but escapes detection in our SDS-containing gels. though there is no reason t,o think that t,his is the case. The existence of a single large amino-terminal domain is suggested by the production of similarly sized stable fragments by a number of proteases (Poteete et aE., 1983). Our results show that residues between 130 and 149 are not essential for the formation of a stable

amino-terminal domain and oligomeric ring, but that they are essential for the formation or stability of a DNA-binding site in the Erf molecule. This is a strong indication that at least part of the DNAbinding site is not an integral part of the aminoterminal domain. Moreover, as discussed above. it’ is likely that this stretch of residues is not an integral part of the carboxy-terminal domain. This line of reasoning leads to the tentative identification of an inter-domain DNA-binding determinant of Erf, that includes at least some of the residues between 130 and 149. The F-130 fragment retains significant affinity for DNA, which leaves unanswered t)he question of whether the amino-terminal domain is directly involved in DNA-binding. Potentially, this question could be addressed by examining amber fragments intermediate in size between F-95 and F-130. Our previous results indicating that an elastase-generated amino-terminal fragment of approximat’ely 136 residues resembles F-149 in its DNA binding affinity further suggests that no residues between 137 and 149 are essential parts of this determinant. There is some uncertainty concerning the location of the carboxy terminus of the elastase fragment. but its mobility in SDS/PAGE indicates a size intermediate between those of F-130 and F-149. The hypothetical int,er-domain DNA-binding determinant is placed between the two domains of Erf in Figure 8. If, as the results described above suggest. there are residues in the middle of the sequence that~ are not located in either domain, then at least some of t,hem must be located between the two domains in the three-dimensional structure. However, not all of them are necessarily so situated. Those tha,t. comprise the DNA-binding determinant, for instance. might reside in a, loop that extends over: and makes contact with, the surface of the amino-terminal domain. The representation in Figure 8 is just the simplest, that is consistent, with our results. A t,wo-domain structural motif is exhibited by a number of ssDNA- or RNA-binding proteins. including the SSB protein of E. coli (Williams et al.. 1983), the gene 32 protein of phage T4 (Hosoda. & Moise, 1978). and the (RNA-binding) rho protein of E. coli (Bear et nl.. 1985). In these and other proteins, t,he amino-terminal domain is responsible for binding to nucleic acid, while the function of the carboxy-terminal domain remains unknown. Proteolytic amino-terminal fragments of both SSB and gene 32 protein have been shown to retain the single-stranded DNA binding affinities of the intact proteins, like the F-149 fragment of Erf. In the cases of both SSB and gene 32 protein, the aminoterminal fragments show an increased ability to destabilize double-stranded DNA relative t,o the intact proteins. This observation has suggested a role for the carboxy-terminal domain in regulating the helix-destabilizing capacity of t’he amino terminal domain. The test for destabilizing doublestranded DNA involves looking for a decrease in the

DNA-binding

117

Determinant in P22 Erf Protein

melting temperature of the DNA in the presence of the protein in question. The apparent sensitivity of domain to thermal Erfs carboxy-terminal denaturation makes such a determination difficult in its case. Evidence from several studies suggests that the carboxy-terminal region of T4 gene 32 protein binds to other proteins in carrying out replication and recombination. The T4 genes 43 (DNA polymerase) and 61 (RNA priming protein) have been shown to bind to gene 32 protein, but not to a proteolytic fragment missing about 60 amino acid residues from the carboxy terminus (Burke et al., 1980, 1985). The analogy with gene 32 protein hints at a similar function for the carboxy-terminal domain of Erf, although there is no direct evidence for this. Genetic studies by Mosig et al. (1977, 1978) have implicated T4 gene 32 protein in interactions with T4 DNA ligase, T4 and E. coli nucleases, T4 topoisomerase, T4 gene 41 priming protein, T4 gene 17 packaging protein and membrane proteins rIIA and rIIB. While most of the mutations responsible for changing these interactions occur in the promoter proximal first-third of the gene, the assignment of these and other functions to known domains remains unclear. Erf, gene 32 protein, and SSB differ strikingly in tertiary structure. Gene 32 protein forms linear polymers of indeterminate length at high concentration (Carroll et al., 1975). SSB consists of stable tetramers in solution (Molineux et al., 1974). Neither of these highly characterized ssDNA binding proteins has been reported to exhibit a revealing morphology in the electron microscope. The shape of Erf makes it possible for us to locate parts of the protein (the 2 domains and the postulated inter-domain DNA-binding determinant) within a definite, albeit low resolution, structure. Interestingly, the transcription termination factor rho has been shown to have a ring-like similar in appearance in oligomeric structure, electron micrographs to that of Erf (Oda & Takanami, 1972). What, if anything, the similarity in domain structure and quaternary organization of Erf and rho implies remains to be determined. This research was supported by grants from the National Institutes of Health, AI18234 to A.R.P. and AI12227 to R.W.H. We thank Larry Hardy for his comments on the manuscript.

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Edited by M. Gottesman