and the single-stranded DNA binding protein of bacteriophage T4

Proc. Nati. Acad. Sci. USA Vol. 84, pp. 8515-8519, December 1987

Genetics

Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4 (zinc finger/helix-destabilizing protein/autoregulation/gene 32 missense mutants/T4 late transcription)

PETER GAUSS, KATHY BOLTREK KRASSA, DAVID S. MCPHEETERS, MARY ANNE NELSON, AND LARRY GOLD Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309

Communicated by Peter H. von Hippel, July 10, 1987 (received for review March 31, 1987)

The DNA binding domain of the gene 32 ABSTRACT protein of the bacteriophage T4 contains a single "zinc-finger" sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. We have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. Our results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression.

Transcription factor IIIA (TFIIIA) of Xenopus laevis binds to a specific sequence in the 5S rRNA genes to activate transcription. TFIIIA contains a sequence, repeated nine times, which has two cysteine and two histidine residues (1). Each TFIIIA molecule binds from 7 to 11 Zn(II) ions (1). Miller et al. (1) proposed that each repeated sequence in TFIIIA binds a single Zn(II) ion and that this structural unit is involved in TFIIIA binding to nucleic acids. Subsequently, several Drosophila proteins and one yeast protein, all thought to be involved in transcriptional regulation, were found to contain a repeated "zinc-finger" motif (2, 3). A single zinc-finger sequence was identified in the gene 32 protein of the bacteriophage T4 (4). Giedroc et al. (5) then demonstrated that the gene 32 protein contains :-1 mol of Zn(II) per mol of protein. The gene 32 protein has been extensively studied as the prototype of proteins that bind to single-stranded nucleic acids in a relatively non-sequence-specific manner (6-8). The protein is required for T4 DNA replication, recombination, and repair (7, 8). It also autogenously regulates its synthesis by competing with ribosomes for the ribosome binding site on its own message (9-13). The central fragment of the gene 32 protein, lacking 21 amino acids from the amino terminus and 50 amino acids from the carboxyl terminus, retains the ability to bind to singlestranded nucleic acids (7, 8). Spectroscopic and chemical modification studies have implicated a region within this central domain, including residues 72-116, in nucleic acid binding (14). This region contains the zinc-finger sequence beginning at residue 77 (4). We isolated a bacterial mutant, Tab32-4, that was unusually restrictive for gene 32 missense mutant phage (15). Tab32-4 was used to isolate a large collection of T4 gene 32 mutants (16). We report here the sequences of these mutant strains. T4 tsL171, the selecting phage for Tab32-4, contains an amino acid change within the presumptive zinc-binding sequence (4). We examined the effect of Zn(II) on all the gene

32 mutants in our collection. The addition of Zn(II) to Tab32-4 cells infected by gene 32 missense mutants suppresses the mutant phage phenotype. Under restrictive conditions, the gene 32 mutations fall into two broad phenotypic classes: the first class is defective in both DNA replication and late transcription, whereas the second class supports high levels of replication but is defective in late transcription.

MATERIALS AND METHODS Media and Chemicals. M9, H-broth, EHA top and bottom agar, and plates were prepared as described (17). Mixed 14C-labeled amino acids were obtained from Schwartz-Mann (catalogue no. 3122-09), and [methyl-3H]thymidine was from Amersham (catalogue no. TRK.418). Strains. Tab32-4 and its parent, NapIV, and all other bacterial strains have been described (15). The T4 phage T4', T4sudl, and the gene 32 mutants amA453, amH18, amHL618, tsP7, tsP401, tsL171, tsG26, mmsl, mms2, and amel are from our stock collection (13, 16). The remaining gene 32 missense mutants were isolated by us (16). Most or all of the newly described gene 32 missense mutants contained an additional uncharacterized mutation (called sudoPam) in the sud locus (16). The precise identity of the sudopam allele is unknown. Sudl is a large deletion of the sud and other genetic loci (18). We constructed 32ts-sud1 and 32ts(sud+) strains for all of the newly isolated gene 32 missense mutants (19). Some mutants were not growth restricted at 37°C in the sud+ background (19). For these mutants, we chose to characterize the strain containing the sudl deletion, so that the effect of zinc addition could be determined at 37°C. Protein and DNA Synthesis after T4 Infection. Proteins made in infected cells were labeled from 5 to 20 min after infection or were pulsed from 25 to 27 min after infection. Preparation of infected cell extracts and electrophoresis were as described (17). [3H]Thymidine incorporation into trichloroacetic acid-precipitable material was measured as described (15). Zinc Addition to Plates and Liquid Infections. T4 was plated as usual (19) except that 0.1 ml of 0.1 M ZnSO4 was added to the 2 ml of top agar before pouring. Each mutant was tested on plates at 42°C and at the lowest restrictive temperature (19). In some cases, to optimize the Zn(II) response, additional temperatures were tested. The infection of liquid cultures was done as described (16), except that zinc sulfate was added to M9 medium in flasks or tubes, either before the infection had begun or at specified times after infection. Sequencing the Mutations Using mRNA as the Template. Synthesis, purification, and labeling of oligonucleotide primers complementary to the gene 32 transcript, mRNA purification, and primer extension RNA sequencing were done as

described (13).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: TFIIIA, transcription factor IIIA. 8515

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RESULTS Mutant Gene 32 Sequences. We sequenced all existing nonsense, missense, and frameshift mutations in gene 32 (ref. 13 and Fig. 1). For most mutants, -150 nucleotides near the genetically mapped site of the mutation were sequenced. Only two mutants (tsl8 and ts74) were completely sequenced; each contains an additional mutation outside the genetically mapped region. Other mutants contain additional nucleotide changes in the region that was sequenced (see Fig. 1). Mutant Infections in Tab32-4 under Normal Growth Conditions. Doherty et al. (16) examined the ability of all gene 32 missense mutants to make plaques on Tab32-4 and NapIV at various temperatures. In general, phage with gene 32 mutations are much less restricted in NapIV cells than in Tab32-4 cells. We examined the effect of gene 32 missense mutations on DNA replication of mutant strains in NapIV and Tab32-4 at 370C. Defects in T4 DNA metabolism can be placed into one of three classes (22): (i) DNA zero-phage that synthesize little or no DNA; (ii) DNA arrest-phage with normal early but defective late replication; and (iii) DNA delayphage with defective early but normal late replication. All three classes of DNA defect are represented in the gene 32 Strain

opC172 op tsRl op tsR2 ts36

ts7O,1 ts56,17,58 tsP7

ts99,5 ts49,85 ts48,90 tsP40 ts53

ts103 ts4CL

tsLl71 ts64 amA453 tsG26 tsl6

amH18 ts74,18 amSR51

ts5l,6 ts52 amSR5lr511 ts92 ts55 ts34 amHL618

ts75

Change

trp(31)>tga trp(31)>arg trp(31) >l eu gly(39)>asp gly(41) >asp al a(43)>thr arg(46)>cys pro(49)>leu ser(50)>phe pro(57)>leu tyr(73)>his his(81)>tyr gly(82)>ser gly(82)>asp pro(88)>leu arg(111)>his trp(116)>tag asp(124)>gly glu(131)>lys gly(132)>ser trp(144) >tag thr(165)>ile cys(166)>tag cys(166)>tyr cys(166)>gln cys(166)>trp pro(167)>ser gly(170)>ser ser(195)>phe

glin(206)>tag

leu(231)>phe

"Zinc-finger" N H2

CYS(77) ser(78) ser(79) thr(80)

HIS(81)

gly(82) asp(83) tyr(84) asp(85) ser(86)

CYS(87) pro(88) val(89) CYS(90)

C02H

FIG. 1. Gene 32 mutant sequences. Five oligonucleotide primers used to sequence all the mutants (13). The amino acid changes corresponding to the nucleotide changes found are shown in the figure. At some mutational sites Doherty et al. (16) identified several isolates that exhibited different phenotypes; for some isolates we found two nucleotide changes. Only the genetically mapped changes are shown. The additional mutations are as follows: ts58, Val-62 to Ala; ts74, Ala-126 to Val; tsl8, Pro-49 to Ser; and ts6, Val-153 to Ile. At other sites, all isolates have a single, identical amino acid change; we assume that at least one isolate has a second change outside of the sequenced region. Four mutations, including tsL171, are located in the suggested Zn(II) binding site, which is shown at the right of the figure. The changes in mutants mmsl, mms2, and ts62 are identical to those of the mutant tsL171. The mutation in amel is a frameshift with the addition of a thymidine to the run of thymidines beginning at nucleotide residue 831. We also include the sequences of tsP7 (20) and amA453 (21). were

mutant collection (19); in addition a few mutations show only minor defects in DNA synthesis. The gene 32 protein binds single-stranded DNA for functions related to DNA metabolism. When no single-stranded DNA is available, the protein binds to gene 32 mRNA, allowing the protein to translationally regulate its own synthesis. Overproduction of the gene 32 protein occurs when an altered protein cannot bind the gene 32 mRNA. Almost all gene 32 missense proteins are overproduced in NapIV at 37TC, labeled from 5 to 20 min after infection (19). Those that are not overproduced at 37TC are overproduced at 42TC (19). Mutant Phage in Tab32-4 Supplemented with Zinc. Berg (4) suggested that the amino acid sequence Cys-(Xaa)3-His(Xaa)5-Cys-(Xaa)2-Cys may be a metal binding domain in the gene 32 protein. Giedroc et al. (5) have confirmed that the protein binds Zn(II), although the exact binding domain remains hypothetical. Within the presumptive Zn(II) binding domain, we identified four gene 32 mutations that are more restricted in the Tab32-4 strain. The mutant, tsL171, with a proline to leucine amino acid change between two potential zinc-ligating cysteine residues, was the very gene 32 mutant used to select the Tab32-4 bacterium (ref. 15 and Fig. 1). We reasoned that perhaps Tab32-4 emerged as a restrictive host for tsL171 because the effective Zn(II) concentration of Tab32-4 was diminished to the point that tsL171 encoded an inactive gene 32 protein at all temperatures. We asked, therefore, if some gene 32 mutations could be suppressed by the addition of Zn(II) to the infected cell. Plaqueformation and burst size. We tested the ability of all gene 32 missense mutants to make plaques on Tab32-4 and NapIV after the addition of Zn(II) to plates. Every mutant responds positively to the addition of zinc to Tab32-4 infections at some temperature (23). For most mutants, at some temperature, the strain does not form plaques without zinc but does form plaques when zinc is added; plaque size varies from very small to the equivalent of wild type. For a few mutants, zinc addition to infections plated at a nonpermissive temperature does not allow plaque formation; however, at a temperature at which extremely tiny plaques are formed, zinc addition increases the size and number of plaques. Most infections of NapIV show no response to zinc (23). A few mutants show a very slight increase in plaque size with zinc addition, but this increase is never comparable to that seen for the same mutant on Tab32-4. Gene 32 amber mutants (amA453, amH18, and amHL618) form no plaques on NapIV or Tab32-4, with or without zinc. They plate equally well on NapIV sul and Tab32-4 sul hosts. Growth is not improved by zinc. Temperature-sensitive mutations in genes 30,41, and 43 are not more restricted in Tab32-4 than in NapIV and are not rescued by zinc at nonpermissive temperatures. For some zinc metalloproteins, one or more other metal ions can be functionally substituted for Zn(II) (24). Co(II), but not Mg(II), Mn(II), Fe(II), Cu(II), or Ca(II), suppresses gene 32 mutant infections in Tab32-4. We also measured the burst size of all gene 32 missense mutants in Tab32-4, with increasing concentrations of Zn(II) from 2 x 1O' M to 2 x 10-" M in M9. Almost all gene 32 missense mutants show a significant increase in burst size (ref. 23 and Table 1). DNA synthesis. For some mutants, we measured the effect of Zn(II) on DNA replication at 37°C (Table 1). The ts5l infection is dramatically responsive to the addition of zinc; a tsSl infection that is DNA negative and yields no viable mature phage becomes essentially wild type in DNA synthesis and burst size (Table 1). tsL171, ts53-sudl, and ts75-sudl in Tab32-4, without Zn(II), make a significant amount of DNA but few viable mature phage. tsL171, in a Tab' bacterium at 42°C, shows a similar phenotype (25). The addition of Zn(II) to Tab32-4 infected with tsL171, ts53-sudl, or ts75-sudl increases phage DNA synthesis slightly but

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Table 1. Effect of added zinc on burst size and DNA synthesis at

370C

Burst size, no. of viable DNA synthesis phage per cell relative to T4+ Tab32-4 NapIV Tab32-4 NapIV + T4 strain Zn Zn Zn + Zn - Zn + Zn Zn + Zn ts53sudl 12 67 57 55 43 72 72 72 tsLl71 3 67 73 125 33 64 86 72 0.5 100 103 123 2 ts5l 79 93 100 ts75sudl 17 94 100 18 40 60 83 79 111 T4+ 82 91 92 92 59 100 86 T4sudl 58 33 23 28 42 42 79 69 Burst size was measured for all gene 32 missense mutants in Tab324 grown in M9 medium with and without added Zn(II). The burst size of almost all mutant infections was increased when zinc was added to the medium (23). Mutants were selected for inclusion in this table based on their relevance to the conclusions drawn from experiments with all the mutants. Burst size was determined as follows. NapIV and Tab32-4 were grown at 370C in M9 medium with no zinc added to 3 x 108 cells per ml. Cells were infected at a multiplicity of infection of 10. At S min after infection, 10-pl aliquots of each culture were diluted into tubes containing 0.5 ml of M9 medium with no added zinc and with zinc added to a final concentration of 2 x 10-7 M, 2 x 10-6 M, 2 x 10-5 M, or2 x 10-4 M. At 90 min after infection, cells were lysed with chloroform, and viable phage titers were determined on NapIV at 30°C. Burst sizes are given as the number of viable progeny per cell for infections with no zinc added (-) and with zinc added to 2 x 10-1 M (+). Although the optimal Zn(II) coflcentration varied from mutant to mutant, if there was an increase in burst size it was demonstrable at 2 x 10-5 M Zn(II). DNA synthesis was measured as follows. Infections were as described for burst size, except that 2 x 10-1 M zinc was added to flasks before infection. Labeling with [3H]thymidine was initiated 5 min after infection by the addition of 1-rnl aliquots of infected cells to 0.1 ml of labeling mixture (500 ,uCi of [3H]thymidine per ml, 25 ,ug of thymidine per ml, 250 ,ug of deoxyadenosine per ml; 1 Ci = 37 GBq). Incorporation of [3H]thymidine into trichloroacetic acidinsoluble material was measured 60 min after infection. No infected culture incorporated >20o of the added label. The amount of DNA is expressed as the fraction of the amount made in wild-type T4 (T4+) infection of NapIV, which has been adjusted to 100 (in bold numbers).

stimulates a dramatic increase in burst size (Table 1 and Discussion). Autogeny. We tested the effect of 2 x 10-5 M Zn(II) on the gene 32 protein overproduction in Tab32-4 and NapIV cells infected with some gene 32 missense strains at 37°C (ref. 23 and Fig. 2). The relationship of overproduction to viability is

b.

C.

....3

-

.Q 2

-

+

FIG. 2. Gene expression at late times after mutant infection of Tab32-4. Bacteria grown at 370C in M9 medium were infected by T4 strains with (+) and without (-) 2 x 10-5 M zinc. T4D' (a), tsSl (b), and ts53-sudl (c) are shown. After infection, proteins were labeled from 25 to 27 min and separated on 10% polyacrylamide gels. The positions of the gene 32 protein and the gene 23 protein (the major capsid protein) are indicated to the left of the figure.

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complex, varying with phage strain, host, temperature, zinc levels, and time after infection. In general, zinc addition, under conditions in which mutant phage in Tab32-4 are rescued, decreases the overproduction of the gene 32 protein. Late gene expression. The addition of Zn(II) to gene 32 mutant infections of Tab32-4 also has a dramatic effect on the synthesis of proteins controlled by late promoters. This effect on late protein synthesis is illustrated by the pattern of synthesis of gene 23 protein, the major structural protein of the T4 capsid (Fig. 2). Gene 32 mutant phage that synthesize large or small amounts of DNA in the absence of Zn(II)such as ts53-sudl and ts5l, respectively-show a similar stimulation of gene 23 synthesis if Zn(II) is added prior to infection (see Discussion). Tab324. The difference in colony size of Tab32-4 and NapIV strains on EHA plates, after growth overnight at 30TC, is striking. Zn(II) or Co(II) addition to plates, at the same concentration that rescues gene 32 missense mutants on Tab32-4, completely reverses the small colony phenotype of Tab32-4 (23); Mg(II), Mn(II), Fe(II), Ca(II), and Cu(II) do not suppress this phenotype. The small colony phenotype is suppressed by the same two divalent cations that rescue gene 32 mutant phage in Tab32-4 cells. This suggests that the effect of Zn(II) or Co(II) on the gene 32 mutation is mediated by some defect in Zn(II) metabolism in the Tab32-4 bacterium.

DISCUSSION The Role of Zn(II) in Gene 32 Protein Function. Berg's prediction (4) that the T4 gene 32 protein contains Zn(II) has been confirmed by Giedroc et al. (5). Our collection of gene 32 mutant phage, isolated as phage unable to grow on Tab32-4 (16), shows a marked positive response to Zn(II) on the restrictive bacterium. The relationship among Tab32-4, the gene 32 mutants, and zinc is understandable if the Tab32-4 defect creates a functionally lower level of Zn(II) and if the mutant proteins have diminished avidity for zinc. The mutations include amino acid substitutions in and around the presumptive zinc finger, as well as amino acid substitutions throughout the core domain of the protein (Fig. 1), suggesting that the entire central domain interacts, directly or indirectly, with zinc. The absence of mutations in the amino- and carboxyl-terminal regions of the protein suggests that these areas do not interact directly with zinc or with the zincbinding domain of the protein. We note that Berg's prediction (4) and the demonstration by Giedroc et al. (5) that the gene 32 protein contains a single Zn(II) ion suggest, but do not prove, that the gene 32 protein has a zinc finger. Although we have assumed that the zinc finger exists, we point out that there is a cluster of mutations (Fig. 1) that surrounds and includes Cys-166, the only cysteine in the gene 32 protein that is not included in the suggested zinc finger. In tsSl this cysteine is changed to a tyrosine. A direct interaction between Cys-166 and Zn(II) must be excluded by experiment. Gene 32 Protein Activates T4 Late Transcription. In bacteriophage T4, ongoing replication is required for late transcription (26, 27). Therefore, all replication proteins are indirectly required for the production of late gene products, and it is difficult to demonstrate that a replication protein is directly required for late transcription. Using special conditions of infection in which a limited amount of late transcription can occur in the absence of replication (28-30), a direct requirement in late transcription for only one replication protein, gp45, has been demonstrated (29, 30). In Tab32-4 cells, gene 32 mutant phage fail to synthesize significant amounts of late gene products (ref. 23 and Fig. 2). For mutants that synthesize no DNA, the lack of late gene products is explained by the obligate coupling of replication and late transcription. However, gene 32 mutants tsS3-sudl,

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tsL171, and ts75-sudl in Tab32-4 make sufficient amounts of DNA to support late transcription (26, 27) but yield few viable mature phage and show reduced amounts of late gene products (ref. 23, Table 1, and Fig. 2). For at least three late genes, the amount of message is greatly reduced for ts53-sudl phage in Tab32-4 cells (23). Addition of 2 x 10' M Zn(II) to these infections restores burst size, autogenous regulation, levels of late gene products, and late message levels to normal, but has only a slight effect on DNA replication (ref. 23, Table 1, and Fig. 2). In tsL171, ts53-sudl, and ts75-sudl infections of Tab32-4, late transcription is uncoupled from replication. Results with these mutants demonstrate a direct requirement for the gene 32 protein in late transcription. The ability of a single-stranded DNA binding protein to facilitate transcription is not unique to the T4 gene 32 protein. The homologous Escherichia coli protein (Ssb) activates promoters transcribed by the bacteriophage N4 virion RNA polymerase. Ssb is required to initiate transcription from specific promoter sites on double-stranded DNA (ref. 31 and L. Rothman-Denes, personal communication). T4 alters the specificity of the host RNA polymerase by substituting T4-encoded gpS5 for the host or factor (32, 33). The T4-modified RNA polymerase (34), containing gp55, initiates transcription at T4 late promoters (32, 33) that contain the consensus -10 region sequence TATAAATA (35). T4 late promoters have no -35 consensus sequence (35, 36). The binding constant (37) for the closed promoter complex on a T4 late promoter is very tight (38), whereas the rate of transition from a closed to an open complex (37) is slow (38). If the limiting step in initiation at T4 late promoters is the transition from closed to open complexes (or the stability of the open complex, if the transition is reversible), one can imagine that gene 32 protein stimulates initiation by binding to the single-stranded DNA of the open complex (37, 39). Stimulation of late transcription by gene 32 protein binding is depicted in Fig. 3. In the simplest version of this stimulation, the T4-modified RNA polymerase forms a closed complex on its own and the

gene 32 protein traps the single-stranded DNA of the open complex by binding nonspecifically to the sugar-phosphate backbone. Since only =10 base pairs of the DNA are unwound in the open promoter complex (39), only one gene 32 monomer binds (7, 8). However, the isolated binding constant of the gene 32 protein to single-stranded DNA is low (7, 8), perhaps too low to activate late transcription. The binding constant of a single gene 32 protein monomer could be significantly increased if the protein binds preferentially to specific structures or sequences present at T4 late promoters. We believe the gene 32 protein can bind preferentially to specific nucleic acid ligands, based on studies of the mRNA target for gene 32 autogenous regulation (13). First, the gene 32 protein binds preferentially to a pseudoknot (defined in ref. 40) in its own message. This preferred binding may result from the unusual constraints imposed on single-stranded RNA loops by the base-paired regions of the pseudoknot, such that the loops of the pseudoknot complement the nucleic acid binding "track" of the gene 32 protein. The structure (if any) of the unwound DNA in an open promoter complex is unknown. An intriguing possibility is that the gene 32 protein binds preferentially to T4 late promoters, because the constrained configuration of the unwound DNA backbone in an initiation complex mimics that of the loop of the pseudoknot on gene 32 mRNA. Second, zinc fingers under study in TFIIIA and other proteins are thought to participate in transcriptional activation by a mechanism involving sequence recognition (1, 3, 41, 42). Two of the three gene 32 mutations that are more deleterious to late transcription than to replication (tsL171 and ts53) are located in the zinc finger, and we suspect that the third mutation (ts75) is located near it in the folded conformation of the protein (J. Hosoda, personal communication). The location of these mutations suggests that the zinc finger may be required for the activation of late transcription and that, by analogy with other zinc fingers, sequence recognition may be involved. The singlestranded region of the operator on gene 32 mRNA (12, 21) consists almost entirely of eight contiguous 5- or 6-base

Sub-Operator cs DNA

VI'ATION ~~~~~~~~~~ACT L-ATE ~~~~~~~OF ~~~~TRAN11SCCRIPTION

S
T4 - Modified RNA Polymerase

FIG. 3. A model for the role of gene 32 in the regulation of late gene expression. To the right are shown the proposed secondary structure and the sequence (as DNA) of the gene 32 mRNA operator. Ovals represent gene 32 protein monomers. After all available sites on single-stranded DNA are filled, the operator becomes the preferred RNA ligand for the gene 32 protein. This binding occludes the translational initiation site and results in repression. The sequence has been organized to emphasize homology with the late promoter consensus sequence (TATAAATA) shown below. Asterisks indicate positions of nonhomology to the promoter. A T4 replication fork is shown to the left. The placement of the late transcription complex on the leading strand is arbitrary. Gene 32 protein binding at the TATAAATA consensus sequence may enhance productive binding by the T4-modified RNA polymerase.

Genetics: Gauss et al. partial repeats of the consensus T4 late promoter sequence (Fig. 3). This single-stranded region, downstream from the nucleating pseudoknot, could be a preferred binding site for the gene 32 protein on RNA (13). Perhaps the gene 32 protein binds preferentially to the late promoter sequence TATAAATA, in the nontemplate strand, thus facilitating transition to an open transcriptional complex. The gene 32 protein has served as a prototype for singlestranded DNA binding proteins and for proteins that autogenously control their own synthesis (6-8). The results and considerations presented here suggest that the gene 32 protein might serve as a prototype for the zinc-finger motif in nucleic acid binding and transcriptional activation. Note Added in Proof. Some preparations of M9 minimal medium contain so little zinc that all the gene 32 mutants exhibit a DO phenotype in Tab32-4. When this happens, we increase the zinc concentration of the medium in small increments to find a zinc level at which tsL171, ts53s, and ts75s (but not ts5l) make a significant amount of DNA and yield few viable progeny phage. In our latest experiments over a narrow range of added ZnSO4 (2-8 ,uM), all the phenotypes reported above for no added ZnSO4 have been confirmed. We thank Tanya Falbel for bringing the Berg (4) paper to our attention, and Mike McCutcheon for help with sequencing. We are grateful to Junko Hosoda, Lucia Rothman-Denes, George Kassavetis, Peter Geiduschek, and Alexander Goldfarb for helpful discussions and for communicating results prior to publication. We are beholden to Kathy Piekarski for her patience and skill in preparing the manuscript. This work was supported by Grant GM19963 from the National Institutes of Health to L.G. 1. Miller, J., McLachlon, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614. 2. Berg, J. (1986) Nature (London) 319, 264-265. 3. Vincent, A. (1986) Nucleic Acids Res. 14, 4385-4391. 4. Berg, J. M. (1986) Science 232, 485-487. 5. Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H. & Coleman, J. E. (1986) Proc. Nati. Acad. Sci. USA 83, 8452-8456. 6. Alberts, B. M. & Frey, L. (1970) Nature (London) 227, 1313-1318. 7. Doherty, D. H., Gauss, P. & Gold, L. (1982) in Multifunctional Proteins: Regulatory and Catalytic/Structural, ed. Kane, J. F. (CRC, Cleveland), pp. 45-72. 8. Kowalczykowski, S. C., Bear, D. G. & von Hippel, P. H. (1981) in The Enzymes, ed. Boyer, P. (Academic, New York), Vol. 14, Part A, pp. 373-444. 9. Russel, M., Gold, L., Morrissett, H. & O'Farrell, P. Z. (1976) J. Biol. Chem. 251, 7263-7270. 10. Krisch, H. M., Bolle, A. & Epstein, R. H. (1974) J. Mol. Biol. 88, 89-104. 11. Lemaire, G., Gold, L. & Yarus, M. (1978) J. Mol. Biol. 126, 73-90. 12. von Hippel, P. H., Kowalczykowski, S. C., Lonberg, N., Newport, J. W., Paul, L. S., Stormo, G. & Gold, L. (1982) J. Mol. Biol. 162, 795-818.

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