Proc. Nati. Acad. Sci. USAVol. 84, pp. 8515-8519, December 1987Genetics

Zinc(II) and the single-stranded DNA binding protein ofbacteriophage 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 GOLDDepartment 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)

ABSTRACT The DNA binding domain of the gene 32protein of the bacteriophage T4 contains a single "zinc-finger"sequence. The gene 32 protein is an extensively studied memberof a class of proteins that bind relatively nonspecifically tosingle-stranded DNA. We have sequenced and characterizedmutations in gene 32 whose defective proteins are activated byincreasing the Zn(II) concentration in the growth medium. Ourresults identify a role for the gene 32 protein in activation of T4late transcription. Several eukaryotic proteins with zinc fingersparticipate in activation of transcription, and the gene 32protein of T4 should provide a simple, well-characterizedsystem in which genetics can be utilized to study the role of azinc finger in nucleic acid binding and gene expression.

Transcription factor IIIA (TFIIIA) ofXenopus laevis binds toa specific sequence in the 5S rRNA genes to activatetranscription. TFIIIA contains a sequence, repeated ninetimes, 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 inTFIIIA binds a single Zn(II) ion and that this structural unitis involved in TFIIIA binding to nucleic acids. Subsequently,several Drosophila proteins and one yeast protein, all thoughtto be involved in transcriptional regulation, were found tocontain a repeated "zinc-finger" motif (2, 3). A singlezinc-finger sequence was identified in the gene 32 protein ofthe bacteriophage T4 (4). Giedroc et al. (5) then demonstratedthat the gene 32 protein contains :-1 mol of Zn(II) per mol ofprotein.The gene 32 protein has been extensively studied as the

prototype of proteins that bind to single-stranded nucleicacids in a relatively non-sequence-specific manner (6-8). Theprotein is required for T4 DNA replication, recombination,and repair (7, 8). It also autogenously regulates its synthesisby competing with ribosomes for the ribosome binding site onits own message (9-13).The central fragment of the gene 32 protein, lacking 21

amino acids from the amino terminus and 50 amino acids fromthe carboxyl terminus, retains the ability to bind to single-stranded nucleic acids (7, 8). Spectroscopic and chemicalmodification studies have implicated a region within thiscentral domain, including residues 72-116, in nucleic acidbinding (14). This region contains the zinc-finger sequencebeginning at residue 77 (4).We isolated a bacterial mutant, Tab32-4, that was unusu-

ally restrictive for gene 32 missense mutant phage (15).Tab32-4 was used to isolate a large collection of T4 gene 32mutants (16). We report here the sequences of these mutantstrains. T4 tsL171, the selecting phage for Tab32-4, containsan amino acid change within the presumptive zinc-bindingsequence (4). We examined the effect of Zn(II) on all the gene

32 mutants in our collection. The addition of Zn(II) toTab32-4 cells infected by gene 32 missense mutants suppres-ses the mutant phage phenotype. Under restrictive condi-tions, the gene 32 mutations fall into two broad phenotypicclasses: the first class is defective in both DNA replicationand late transcription, whereas the second class supportshigh levels of replication but is defective in late transcription.

MATERIALS AND METHODSMedia and Chemicals. M9, H-broth, EHA top and bottom

agar, and plates were prepared as described (17). Mixed14C-labeled amino acids were obtained from Schwartz-Mann(catalogue no. 3122-09), and [methyl-3H]thymidine was fromAmersham (catalogue no. TRK.418).

Strains. Tab32-4 and its parent, NapIV, and all otherbacterial strains have been described (15). The T4 phage T4',T4sudl, and the gene 32 mutants amA453, amH18,amHL618, tsP7, tsP401, tsL171, tsG26, mmsl, mms2, andamel are from our stock collection (13, 16). The remaininggene 32 missense mutants were isolated by us (16). Most orall of the newly described gene 32 missense mutants con-tained an additional uncharacterized mutation (calledsudoPam) in the sud locus (16). The precise identity of thesudopam allele is unknown. Sudl is a large deletion of the sudand other genetic loci (18). We constructed 32ts-sud1 and32ts(sud+) strains for all of the newly isolated gene 32missense mutants (19). Some mutants were not growthrestricted at 37°C in the sud+ background (19). For thesemutants, we chose to characterize the strain containing thesudl deletion, so that the effect of zinc addition could bedetermined at 37°C.

Protein and DNA Synthesis after T4 Infection. Proteinsmade in infected cells were labeled from 5 to 20 min afterinfection or were pulsed from 25 to 27 min after infection.Preparation ofinfected cell extracts and electrophoresis wereas described (17). [3H]Thymidine incorporation into tri-chloroacetic acid-precipitable material was measured asdescribed (15).

Zinc Addition to Plates and Liquid Infections. T4 was platedas usual (19) except that 0.1 ml of 0.1 M ZnSO4 was added tothe 2 ml of top agar before pouring. Each mutant was testedon plates at 42°C and at the lowest restrictive temperature(19). In some cases, to optimize the Zn(II) response, addi-tional temperatures were tested. The infection of liquidcultures was done as described (16), except that zinc sulfatewas added to M9 medium in flasks or tubes, either before theinfection had begun or at specified times after infection.Sequencing the Mutations Using mRNA as the Template.

Synthesis, purification, and labeling of oligonucleotide prim-ers complementary to the gene 32 transcript, mRNA purifi-cation, and primer extension RNA sequencing were done asdescribed (13).

Abbreviation: TFIIIA, transcription factor IIIA.

8515

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Proc. Natl. Acad. Sci. USA 84 (1987)

RESULTS

Mutant Gene 32 Sequences. We sequenced all existingnonsense, missense, and frameshift mutations in gene 32 (ref.13 and Fig. 1). For most mutants, -150 nucleotides near thegenetically mapped site of the mutation were sequenced.Only two mutants (tsl8 and ts74) were completely se-quenced; each contains an additional mutation outside thegenetically mapped region. Other mutants contain additionalnucleotide changes in the region that was sequenced (see Fig.1).Mutant Infections in Tab32-4 under Normal Growth Con-

ditions. Doherty et al. (16) examined the ability of all gene 32missense mutants to make plaques on Tab32-4 and NapIV atvarious temperatures. In general, phage with gene 32 muta-tions are much less restricted in NapIV cells than in Tab32-4cells. We examined the effect of gene 32 missense mutationson DNA replication of mutant strains in NapIV and Tab32-4at 370C. Defects in T4 DNA metabolism can be placed intoone of three classes (22): (i) DNA zero-phage that synthe-size little or no DNA; (ii) DNA arrest-phage with normalearly but defective late replication; and (iii) DNA delay-phage with defective early but normal late replication. Allthree classes of DNA defect are represented in the gene 32

Strain

opC172op tsRlop tsR2ts36ts7O,1ts56,17,58tsP7ts99,5ts49,85ts48,90tsP40ts53ts103ts4CLtsLl71ts64amA453tsG26tsl6

amH18ts74,18amSR51ts5l,6ts52amSR5lr511ts92ts55ts34amHL618ts75

Change "Zinc-finger"

trp(31)>tga NH2trp(31)>argtrp(31) >l eugly(39)>aspgly(41) >aspal a(43)>thrarg(46)>cys CYS(77)pro(49)>leu ser(78)ser(50)>phe ser(79)pro(57)>leu thr(80)tyr(73)>his HIS(81)his(81)>tyr gly(82)gly(82)>ser asp(83)gly(82)>asp tyr(84)pro(88)>leu asp(85)arg(111)>his ser(86)trp(116)>tag CYS(87)asp(124)>gly pro(88)glu(131)>lys val(89)gly(132)>ser CYS(90)trp(144) >tagthr(165)>ilecys(166)>tagcys(166)>tyrcys(166)>glncys(166)>trppro(167)>sergly(170)>serser(195)>pheglin(206)>tag C02Hleu(231)>phe

FIG. 1. Gene 32 mutant sequences. Five oligonucleotide primerswere used to sequence all the mutants (13). The amino acid changescorresponding to the nucleotide changes found are shown in thefigure. At some mutational sites Doherty et al. (16) identified severalisolates that exhibited different phenotypes; for some isolates wefound two nucleotide changes. Only the genetically mapped changesare shown. The additional mutations are as follows: ts58, Val-62 toAla; 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 thesequenced region. Four mutations, including tsL171, are located inthe suggested Zn(II) binding site, which is shown at the right of thefigure. The changes in mutants mmsl, mms2, and ts62 are identicalto those of the mutant tsL171. The mutation in amel is a frameshiftwith the addition of a thymidine to the run of thymidines beginningat nucleotide residue 831. We also include the sequences of tsP7 (20)and amA453 (21).

mutant collection (19); in addition a few mutations show onlyminor defects in DNA synthesis.The gene 32 protein binds single-stranded DNA for func-

tions related to DNA metabolism. When no single-strandedDNA is available, the protein binds to gene 32 mRNA,allowing the protein to translationally regulate its own syn-thesis. Overproduction of the gene 32 protein occurs when analtered protein cannot bind the gene 32 mRNA. Almost allgene 32 missense proteins are overproduced in NapIV at37TC, labeled from 5 to 20 min after infection (19). Those thatare 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 thegene 32 protein. Giedroc et al. (5) have confirmed that theprotein binds Zn(II), although the exact binding domainremains hypothetical. Within the presumptive Zn(II) bindingdomain, we identified four gene 32 mutations that are morerestricted in the Tab32-4 strain. The mutant, tsL171, with aproline to leucine amino acid change between two potentialzinc-ligating cysteine residues, was the very gene 32 mutantused to select the Tab32-4 bacterium (ref. 15 and Fig. 1). Wereasoned that perhaps Tab32-4 emerged as a restrictive hostfor tsL171 because the effective Zn(II) concentration ofTab32-4 was diminished to the point that tsL171 encoded aninactive gene 32 protein at all temperatures. We asked,therefore, if some gene 32 mutations could be suppressed bythe 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 andNapIV after the addition of Zn(II) to plates. Every mutantresponds positively to the addition of zinc to Tab32-4 infec-tions at some temperature (23). For most mutants, at sometemperature, the strain does not form plaques without zincbut does form plaques when zinc is added; plaque size variesfrom very small to the equivalent of wild type. For a fewmutants, zinc addition to infections plated at a nonpermissivetemperature does not allow plaque formation; however, at atemperature at which extremely tiny plaques are formed, zincaddition increases the size and number of plaques. Mostinfections of NapIV show no response to zinc (23). A fewmutants show a very slight increase in plaque size with zincaddition, but this increase is never comparable to that seenfor the same mutant on Tab32-4. Gene 32 amber mutants(amA453, amH18, and amHL618) form no plaques on NapIVor Tab32-4, with or without zinc. They plate equally well onNapIV sul and Tab32-4 sul hosts. Growth is not improved byzinc. Temperature-sensitive mutations in genes 30,41, and 43are not more restricted in Tab32-4 than in NapIV and are notrescued by zinc at nonpermissive temperatures. For somezinc metalloproteins, one or more other metal ions can befunctionally substituted for Zn(II) (24). Co(II), but notMg(II), Mn(II), Fe(II), Cu(II), or Ca(II), suppresses gene 32mutant 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 32missense 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 ts5linfection is dramatically responsive to the addition of zinc; atsSl infection that is DNA negative and yields no viablemature phage becomes essentially wild type in DNA synthe-sis and burst size (Table 1). tsL171, ts53-sudl, and ts75-sudlin Tab32-4, without Zn(II), make a significant amount ofDNA but few viable mature phage. tsL171, in a Tab'bacterium at 42°C, shows a similar phenotype (25). Theaddition of Zn(II) to Tab32-4 infected with tsL171, ts53-sudl,or ts75-sudl increases phage DNA synthesis slightly but

8516 Genetics: Gauss et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 8517

Table 1.at 370C

Effect of added zinc on burst size and DNA synthesis

Burst size, no. of viable DNA synthesisphage 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 43 55 72 72 72tsLl71 3 67 73 125 33 64 86 72ts5l 0.5 100 103 123 2 79 93 100ts75sudl 17 94 100 18 40 60 83 79T4+ 82 111 91 92 92 59 100 86T4sudl 58 23 33 28 42 42 79 69

Burst size was measured for all gene 32 missense mutants inTab324 grown in M9 medium with and without added Zn(II). Theburst size of almost all mutant infections was increased when zincwas added to the medium (23). Mutants were selected for inclusionin this table based on their relevance to the conclusions drawn fromexperiments with all the mutants. Burst size was determined asfollows. NapIV and Tab32-4 were grown at 370C in M9 medium withno zinc added to 3 x 108 cells per ml. Cells were infected at amultiplicity of infection of 10. At S min after infection, 10-pl aliquotsof each culture were diluted into tubes containing 0.5 ml of M9medium with no added zinc and with zinc added to a final concen-tration of 2 x 10-7 M, 2 x 10-6 M, 2 x 10-5 M, or2 x 10-4 M. At90 min after infection, cells were lysed with chloroform, and viablephage titers were determined on NapIV at 30°C. Burst sizes are givenas the number of viable progeny per cell for infections with no zincadded (-) and with zinc added to 2 x 10-1 M (+). Although theoptimal Zn(II) coflcentration varied from mutant to mutant, if therewas an increase in burst size it was demonstrable at 2 x 10-5 MZn(II). DNA synthesis was measured as follows. Infections were asdescribed for burst size, except that 2 x 10-1 M zinc was added toflasks before infection. Labeling with [3H]thymidine was initiated 5min after infection by the addition of 1-rnl aliquots of infected cellsto 0.1 ml of labeling mixture (500 ,uCi of [3H]thymidine per ml, 25 ,ugof thymidine per ml, 250 ,ug of deoxyadenosine per ml; 1 Ci = 37GBq). Incorporation of [3H]thymidine into trichloroacetic acid-insoluble material was measured 60 min after infection. No infectedculture incorporated >20o of the added label. The amount of DNAis expressed as the fraction of the amount made in wild-type T4 (T4+)infection of NapIV, which has been adjusted to 100 (in boldnumbers).

stimulates a dramatic increase in burst size (Table 1 andDiscussion).Autogeny. We tested the effect of 2 x 10-5 M Zn(II) on the

gene 32 protein overproduction in Tab32-4 and NapIV cellsinfected with some gene 32 missense strains at 37°C (ref. 23and Fig. 2). The relationship of overproduction to viability is

....3

2

- +

b.

-

.Q

C.

FIG. 2. Gene expression at late times after mutant infection ofTab32-4. Bacteria grown at 370C in M9 medium were infected by T4strains with (+) and without (-) 2 x 10-5 M zinc. T4D' (a), tsSl (b),and ts53-sudl (c) are shown. After infection, proteins were labeledfrom 25 to 27 min and separated on 10% polyacrylamide gels. Thepositions of the gene 32 protein and the gene 23 protein (the majorcapsid protein) are indicated to the left of the figure.

complex, varying with phage strain, host, temperature, zinclevels, and time after infection. In general, zinc addition,under conditions in which mutant phage in Tab32-4 arerescued, decreases the overproduction ofthe gene 32 protein.Late gene expression. The addition of Zn(II) to gene 32

mutant infections ofTab32-4 also has a dramatic effect on thesynthesis ofproteins controlled by late promoters. This effecton late protein synthesis is illustrated by the pattern ofsynthesis of gene 23 protein, the major structural protein ofthe T4 capsid (Fig. 2). Gene 32 mutant phage that synthesizelarge or small amounts of DNA in the absence of Zn(II)-such as ts53-sudl and ts5l, respectively-show a similarstimulation of gene 23 synthesis if Zn(II) is added prior toinfection (see Discussion).

Tab324. The difference in colony size of Tab32-4 andNapIV strains on EHA plates, after growth overnight at 30TC,is striking. Zn(II) or Co(II) addition to plates, at the sameconcentration that rescues gene 32 missense mutants onTab32-4, completely reverses the small colony phenotype ofTab32-4 (23); Mg(II), Mn(II), Fe(II), Ca(II), and Cu(II) do notsuppress this phenotype. The small colony phenotype issuppressed by the same two divalent cations that rescue gene32 mutant phage in Tab32-4 cells. This suggests that the effectof Zn(II) or Co(II) on the gene 32 mutation is mediated bysome defect in Zn(II) metabolism in the Tab32-4 bacterium.

DISCUSSIONThe Role of Zn(II) in Gene 32 Protein Function. Berg's

prediction (4) that the T4 gene 32 protein contains Zn(II) hasbeen confirmed by Giedroc et al. (5). Our collection of gene32 mutant phage, isolated as phage unable to grow on Tab32-4(16), shows a marked positive response to Zn(II) on therestrictive bacterium. The relationship among Tab32-4, thegene 32 mutants, and zinc is understandable if the Tab32-4defect creates a functionally lower level of Zn(II) and if themutant proteins have diminished avidity for zinc. The mu-tations include amino acid substitutions in and around thepresumptive zinc finger, as well as amino acid substitutionsthroughout the core domain of the protein (Fig. 1), suggestingthat the entire central domain interacts, directly or indirectly,with zinc. The absence of mutations in the amino- andcarboxyl-terminal regions of the protein suggests that theseareas do not interact directly with zinc or with the zinc-binding 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 singleZn(II) ion suggest, but do not prove, that the gene 32 proteinhas a zinc finger. Although we have assumed that the zincfinger exists, we point out that there is a cluster of mutations(Fig. 1) that surrounds and includes Cys-166, the onlycysteine in the gene 32 protein that is not included in thesuggested zinc finger. In tsSl this cysteine is changed to atyrosine. A direct interaction between Cys-166 and Zn(II)must be excluded by experiment.Gene 32 Protein Activates T4 Late Transcription. In bacte-

riophage T4, ongoing replication is required for late tran-scription (26, 27). Therefore, all replication proteins areindirectly required for the production of late gene products,and it is difficult to demonstrate that a replication protein isdirectly required for late transcription. Using special condi-tions of infection in which a limited amount of late transcrip-tion can occur in the absence of replication (28-30), a directrequirement in late transcription for only one replicationprotein, gp45, has been demonstrated (29, 30).

In Tab32-4 cells, gene 32 mutant phage fail to synthesizesignificant amounts of late gene products (ref. 23 and Fig. 2).For mutants that synthesize no DNA, the lack of late geneproducts is explained by the obligate coupling of replicationand late transcription. However, gene 32 mutants tsS3-sudl,

Genetics: Gauss et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

tsL171, and ts75-sudl in Tab32-4 make sufficient amounts ofDNA to support late transcription (26, 27) but yield few viablemature phage and show reduced amounts of late geneproducts (ref. 23, Table 1, and Fig. 2). For at least three lategenes, the amount of message is greatly reduced for ts53-sudlphage in Tab32-4 cells (23). Addition of 2 x 10' M Zn(II) tothese infections restores burst size, autogenous regulation,levels of late gene products, and late message levels tonormal, but has only a slight effect on DNA replication (ref.23, Table 1, and Fig. 2). In tsL171, ts53-sudl, and ts75-sudlinfections of Tab32-4, late transcription is uncoupled fromreplication. Results with these mutants demonstrate a directrequirement 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) activatespromoters transcribed by the bacteriophage N4 virion RNApolymerase. Ssb is required to initiate transcription fromspecific promoter sites on double-stranded DNA (ref. 31 andL. 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) thatcontain the consensus -10 region sequence TATAAATA(35). T4 late promoters have no -35 consensus sequence (35,36). The binding constant (37) for the closed promotercomplex on a T4 late promoter is very tight (38), whereas therate of transition from a closed to an open complex (37) isslow (38). If the limiting step in initiation at T4 late promotersis the transition from closed to open complexes (or thestability of the open complex, if the transition is reversible),one can imagine that gene 32 protein stimulates initiation bybinding to the single-stranded DNA of the open complex (37,39). Stimulation of late transcription by gene 32 proteinbinding is depicted in Fig. 3.

In the simplest version of this stimulation, the T4-modifiedRNA polymerase forms a closed complex on its own and the

gene 32 protein traps the single-stranded DNA of the opencomplex by binding nonspecifically to the sugar-phosphatebackbone. Since only =10 base pairs of the DNA areunwound in the open promoter complex (39), only one gene32 monomer binds (7, 8). However, the isolated bindingconstant of the gene 32 protein to single-stranded DNA is low(7, 8), perhaps too low to activate late transcription. Thebinding constant of a single gene 32 protein monomer couldbe significantly increased if the protein binds preferentially tospecific 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 mRNAtarget for gene 32 autogenous regulation (13). First, the gene32 protein binds preferentially to a pseudoknot (defined inref. 40) in its own message. This preferred binding may resultfrom the unusual constraints imposed on single-strandedRNA loops by the base-paired regions of the pseudoknot,such that the loops of the pseudoknot complement the nucleicacid binding "track" of the gene 32 protein. The structure (ifany) of the unwound DNA in an open promoter complex isunknown. An intriguing possibility is that the gene 32 proteinbinds preferentially to T4 late promoters, because the con-strained configuration of the unwound DNA backbone in aninitiation complex mimics that of the loop of the pseudoknoton gene 32 mRNA. Second, zinc fingers under study inTFIIIA and other proteins are thought to participate intranscriptional activation by a mechanism involving se-quence recognition (1, 3, 41, 42). Two of the three gene 32mutations that are more deleterious to late transcription thanto replication (tsL171 and ts53) are located in the zinc finger,and we suspect that the third mutation (ts75) is located nearit in the folded conformation of the protein (J. Hosoda,personal communication). The location of these mutationssuggests that the zinc finger may be required for the activa-tion of late transcription and that, by analogy with other zincfingers, sequence recognition may be involved. The single-stranded region of the operator on gene 32 mRNA (12, 21)consists almost entirely of eight contiguous 5- or 6-base

S ~~~~~~~~~~ACTVI

< ~~~~~~~OFL

< ~~~~TRAN11SC

Gene 32 Protein

<! \ T4 - ModifiedRNA Polymerase

Sub-Operator cs DNA

TTAATT AAATTAA ATTA AAAAGG A AATAA AAAT G TTTAA CGT AAT

T4 LATEPROMOTER CONSENSUS:

TATAAATA

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 structureand the sequence (as DNA) of the gene 32 mRNA operator. Ovals represent gene 32 protein monomers. After all available sites on single-strandedDNA are filled, the operator becomes the preferred RNA ligand for the gene 32 protein. This binding occludes the translational initiation siteand 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 thelate transcription complex on the leading strand is arbitrary. Gene 32 protein binding at the TATAAATA consensus sequence may enhanceproductive binding by the T4-modified RNA polymerase.

8518 Genetics: Gauss et al.

'ATION-ATECRIPTION

Proc. NaMl. Acad. Sci. USA 84 (1987) 8519

partial repeats of the consensus T4 late promoter sequence(Fig. 3). This single-stranded region, downstream from thenucleating pseudoknot, could be a preferred binding site forthe gene 32 protein on RNA (13). Perhaps the gene 32 proteinbinds preferentially to the late promoter sequence TATAA-ATA, in the nontemplate strand, thus facilitating transition toan open transcriptional complex.The gene 32 protein has served as a prototype for single-

stranded DNA binding proteins and for proteins that auto-genously control their own synthesis (6-8). The results andconsiderations presented here suggest that the gene 32protein might serve as a prototype for the zinc-finger motif innucleic acid binding and transcriptional activation.

Note Added in Proof. Some preparations of M9 minimal mediumcontain so little zinc that all the gene 32 mutants exhibit a DOphenotype in Tab32-4. When this happens, we increase the zincconcentration of the medium in small increments to find a zinc levelat which tsL171, ts53s, and ts75s (but not ts5l) make a significantamount of DNA and yield few viable progeny phage. In our latestexperiments over a narrow range of added ZnSO4 (2-8 ,uM), all thephenotypes reported above for no added ZnSO4 have been con-firmed.

We thank Tanya Falbel for bringing the Berg (4) paper to ourattention, and Mike McCutcheon for help with sequencing. We aregrateful to Junko Hosoda, Lucia Rothman-Denes, George Kas-savetis, Peter Geiduschek, and Alexander Goldfarb for helpfuldiscussions and for communicating results prior to publication. Weare beholden to Kathy Piekarski for her patience and skill inpreparing the manuscript. This work was supported by GrantGM19963 from the National Institutes of Health to L.G.

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