J. Nol.

Bid.

(1979) 128, 2147

Structural Organization of Escherichia coli tRNATYr Gene Clusters in Four Different Transducing Bacteriophages tI~~-~~

?J.Rossr,

\YII,MA

Ross,

JAMES

,%RTHl:R

&AK,

DAVII)

,J.

LIPMA?:

ANL)

LAiW-i-

transfer ‘I‘t~e struclnral organizatimt of ttw gwlrs coding for tyrosillc-accepting RNA in Escherichia coli has been studied by restriction rnzyrnr analysis and DNA scq1lencing, utilizing four different specialized transducing bacteriophages. Tlw non-defecti\-e transducing ptlage, +8Ol~s~I~i- (doublet), carries two 85 base-pair sequences corresponding to mature tRNA:Fr, separated by a 2OWbasepair “spacer”. A derivative of ttlis pllagc, +3Opsu~, (singlet), has lost one of the t’wo W-base-pair tRNAT” sequences alld ttle X)()-base-pair spacer. The sequences upstream and downstream of the t,RNriT!“’ mature structural sequence are identical in bottl phages. Tile pent’ codin g for tRNAp has been studied using tuo specialized transduk~~g ptlages of comptrtely different origin from eacll ot,hc-r, XtxEN)dc$yTsu:& and hrifd 18. A portion of t 110 E. coli DNA carried by tttew two phages is identical: t,tlree tRNA pews are clustered in a region of approximately 350 base-pairs wit,11 the arranyerneIlt, and orientation (relative t’o transcription) 5’ . . . tRNA~~“-tKNA~‘“-tR?iATh’. l’tw sequences corresponding to IllattIre tRNAzlY and tRN.4;“’ are separated from each otllrr by six base-pairs 100 base-pairs. and from tRNAF’ by approximately Surprisingly, sequences upstream of the tRNAFr gclle are completely different. ill the two transducing phayes, althougll scc~ue~~cc~sincluding, and downstream of, tlrcl tRNAS” gene arv ttlr same. Tile reyiotl immediately upstream of tllc tRNAS7’ gene on Ah80dglyTsu,& is identical, or trrarly identical, for at 1eAst 500 basc~-pairs to the corresponding region upstrealn of t,RNATY’ in the @Opsu;l;(doublet) a,nd +8Opsui, (singlet) transdllcing phages. In cont’rast, the E. col% upstream of tlw DNA it, hri,fd18 has an adtlit,ional tRX:A gellrx immediakly t.RNAT” gene, and there is littlo or 1~ I~omology wittl the upstream sectue?xrs of arly of the other phaptls. It thus appears ttlat ttle regions surrounding anct irlcludinp ttlr tRN4TY’ g,renrh on hh8OdglyTsu$ art’ a Il>.brid of tRNA’F” upstrwm *Iw do\\~nstream src,“t’nc’s. ;~rrd tRNA~-tRNA;‘r-tRNAE+ All of ttle five tRNA gclws studied at-v cotinwr with the kr~owr~ scyw~lcen for t twir respective mature tKNAs. This rules ol~t ttlv possihility of iusert,ed sectuenws \\ ittrin those coding for ttlcs nlaturo tKNAs, whictl :IW rwnoved during processing to a matrwe tRNA. The implications of these tRNB gcnc strllctural studies for extrapolation back ils well as ttrc, wla tionsh ips t~ctween gene structuw to the E. co/i chromosome, wld transcriptional analyses. arc disclwxtl. t To whom correspondence

0022 -%836/79/050021-27

shoultt he sent.

$02.00/O

I(_‘ 1979 .~cdenxc:

Prtws

Inc.

(London)

Ltd.

.,., I-

.I. .r. ROSYI E[II .4 I,.

1. Introduction In Escherichia coli three genes that cotlt~ for two dist’inct species of transfer RNA tyrosine (tRXATY’) have been identified (Garen ef al., 1965; Signer et a&, 1965: Goodman et al., 1968; Russell et al.. 1970: Orias et al., 1972; Bachmann et al., 1976). A single gene, tyrL’ at 88 minutes, codes for tRNAzY” (Eggersten & Adelberg, 1965: Orias et al., 1972: Squires et ul.. 1973: Bachmann et al., 1976) and is in close proximity to the gZyT, thrT genes t,hat code for tRNAzlY and tRNAy, respectively (Orias et al., 1972; Squires et ab.. 1973; Bachmann et al.. 1976: Ikemura & Ozeki, 1977). The tyrT locus at 27 minutes is composed of two genes which code for tRNA;PY’ (Garcn et al., 1965: Signer et al., 1965: Russell et al., 1970: Bachmann et al.. 1976). The nucleotide sequences of tRNAy” and tRNATY’ differ from each other by only two bases in the variable loop region (Goodman et al.. 1968.1970). but, the functional significance of this difference is not knobvn. Studies on t,yrosine-accepting activity during early logarithmic indicate nearly equal amounts of tRNAy’ and tRNAF’ during late log phase growth, but, a twofold excess of tRNAz”‘- accepting activity (Gross $ Raab. 1972). The mechanism(s) responsible for this changing ratio of tyrosine-accepting activity are not understood. but. they may reflect. differences either in the regulation of t,ranscript,ion from the t)RNAF“ and t,RNATY’ genes or in of the two t’RNATyr spt&s. the IJrOCeSSing A great deal of information concerning transcription and processing of tRiYA gene clusters has come from analyses ut,ilizing specialized t,ransducing bact,eriophages carrying one or more tRNA genes (for a review. see Brookhaven Syn~p. Biol., 1974). Transducing phages carrying either the tRNAF’ or the tRNA~‘r-tRN-~~l’-tRNA~’ cluster have been extensively ut,ilized in studies. both it? viva and irr vitro. of tRNA transcription and processing (Russell et trl.. 1970; Squires et al., 1973: ,Altman et al., 1974; Carbon et al., 1974; Alt,man, 1975: Chang & Carbon. 1975; Daniel et a.Z., 1975; Schedl et ul.? 1976: Smith, 1976: Grimberg & Daniel, 1977). Rest’riction endonuclease fragments isolated from t,hese tsransducing phagr have also been very useful in studying t,he initiat)ion (Kiipper it nl.. 1975>1976) and termination (Kiipper st al.. 1978) of tRNA gene transcription. phagc One of the st,riking results emerging from studies with t)hese transducing was the finding that, the 59 base-pairs that immediat’ely precede the startpoint of transcription in the tRNA, Tyl’ gene. carried on +8Opsu&. have an identical sequence t.o the tRNATY’ gene, carried on Xh80dgZyTsu& (Sekiya et al., 1976). This result, the only comparison available for tRNA promoter regions, strongly suggested similar, if not identical, regulatory sequences for tRNA genes on opposite sides of the chromosome. Since it was known that the tRNA~r-tRNA~lY-tRNA~r genes are clustered within a region of approximately 500 base-pairs on Xh80dglyTsu,f, (Wu et al., 1973), it was suggested that the three t’RN&ds might be cotranscribed as a polycistronic precursor (Squires et al.. 1973). However, transcriptional studies in viva and in vitro (Chang & Carbon, 1975; Daniel et nl., 1975: llgen et al., 1976; Grimberg & Daniel, 1977) present conflicting evidence concerning this question, which until now was further complicated by not. knowing the relative order of these three tRNA genes. At least two specialized transducing phages carrying the gene for tRNA2’ have been isolated : hh80dgZyTs& (Squires et t&Z..1973) and Xrifd18 (Kirschbaum & Konrad, 1973). The experiments reported here start with the isolation of a HinIl 1070-basepair fragment, from hh80dgZyTsu,f, and a 11X1 2700-base-pair fragment from

0Rc:AXIZ.~TI0S

OF’ tRr\::\T”r

(:ESE

(‘1,1~S’l’ERS

23

Xrifd18, each of which carries the tRNA genes tyr[‘, gZyT and thrT. III earlier experiments Landy et al. (1974a) isolated a H&II f III 730-base-pair fragment from fragment from $8Opsu$ - (doublet 48Opsu:,, (singlet phage) and a 1020-base-pair phage) that carry one or two copies, respectively. of the sequence for tRNAy’. As one of t’he initial steps in studying regulation of expression of the tRNATY’ and tRNA~r-tJRNA~l’-tRNA~r gene clusters. the structure and organization of legulat’orp and coding sequences for these genes on four different t#ransducing phages have been det)ermined and compared. Surprisingly, t’lir seyurncrs upstream of t hcl st’art’point, of transcription of tRNAF” and tRKATY” carried on $80psu1~, (singlet), $8Opsu:,; -- (doublet,), and hh8OdgZyTsu& are shon~r t,o be ident#ical, or near identical. The region upstream of t’R?u’Ap” on hrl,fd18 does not for at least 500 base-pairs. display this homology. The genes for tRh’A7’. tRNAz”, and t,RNA$’ are clusteretl wit~hin a sequence of 350 base-pairs wit’h the orientatjion 5’-tRNAy’-t’RNAf’“tRNX$” on both /\h80dgZ@u& and hrifd18. These results suggest either two separak genet,ic locabions in E. coli for t)his t,RNA gc’nc’ cluster or t hc gencration of IKW l,egulat’o~~-stnlctural combinations in thtl form&ion of one or bot’h of the tRPU’Ay”tRNAGly-t,RNAThP transducing phages. Furthermore. tjhcb distance bet’ween t hc tRNA& gent’ kid thfa tRNA~‘Y-t~RNA~h” genes was fount1 to be about 100 has+ pairs. \z,hich is more than sufficirnt for a promot’er bctwtben these genes. The resuks presented also confirm that the onl,v structural differences between the tRXAT”” “singlet’” and “doublet” phages are thtt ahsencr of t#he intergenic spacer sequence and the second, or SLI , copy of tRNAy’ In addibion to the above results, it, is thou-n t,hat an unusual series of l’i%base-pair tandtxmly repeated sequenacxs beginning within t,he tRNA-\T”” mat’ure pent scqurnce (Span & Landy, 1978) and sitcx (liiippt~r of r/l.. I978) arc aknt front cant aining a rho-dependent tc,rminatiotr t hcb t ESSAY’.

g:1~notic~ rt@on.

2. Materials and Methods (a) liacterial

and bacteriophage

strains

and

rnicrohiological

procedures

+8Opsu& (singlet) and +8OpsclGi- (doubk%) (Russell et al., 1970) were used as the sources of tRNATYr DNA and \v~e prepared according to Landy et al. (1974~). For thfx t-KNAT”‘-tRNA;“-tRNArh’ gene cluster, 2 bacterioptlages were employed, Xc1857S7r$d18 (Kirschbaum bt Konrad, 1973) (referred t.o llere as hrgd18), and hcI857S7.h8OdglyTsu& (Squires et a/., 1973) (referred to here as Ah80dgZyTsu,&). hrifd18 was prepared from a heat-inducible lysogen of E. coli CA274 (trlj-am, Zac -am. Xrijd18, hcI857S7, x:is6) (supplied by M. Nomura) according to t~llt~ prot,ocol of Kirsctlbaum & Konrad ( 1973). Xh8OdglyTsuA was prepared from the 1~eat-indllciblc~ 1ysoge11 .I(‘1 00 (trpL4,,, argH. lacZUAA,; hh8UdglylTsu.$. AcI857S7.1180) (supplied t)y .I. (‘arbors) according to the following procedure. An overnight inoculum iI1 Casamino acids medium (W5‘!,) WMS diluted 1: 10 in 2 \\ LB (2% tryptone, 1% yeast extract,, 2oi;, NaCl) and prolvn with vigorous aeration for several hours to an A,,, of 1.7 to 1.8 O.U. lmits. AII equal volume of 1 x LB, heated to 55°C’ was added. and the culture was incubated at, 42°C for 15 min, then quickly chilled to 31°C. incubated wit)h moderate aeration for an additional I.5 h. and then vigorous aeration for a final O-5 II. Cells were harvested by cerrtrifugattion. resuspended in l/60 vol. of 10 mM-Tris.HCl (pH 7.4), 5 mM-MgSO, buffer and frozen at --20°C. Cells were lysed by thawing to room temperature followed by addition of 0.1 vol. chloroform and shaking at, 37°C for O-5 h. To this suspension, DNAase and lysozyme were added to final concentrations of 10 pg/ml and 200 @g/ml, respectively. For all bacteriophage preparations, phnp~ were purified by CsCl step gradient)s. with steps of I.3 (containing phage), 1.4. 1.5

DNA4 was prcparcd from Itactcrioplrag~~ pwparatiotts accorditlg to Latltly et al. ( 1974~:). All wstrictiott cwdottttclrasc: tligestiotts, cxwpt~ EcoHI wcrr-Tris.HCl (pH 7.5). 5 tnht-MyCl,, 50 tnw NaC!l. Tlte digestiott conditions \v(:r( 30 to 100 pg I)NA irtll and sttficicttt, clnzytw (as prerionsly dc~twtnitic~tl 1)~ titratiott) to proditw a litnit dipcast iott it1 tltcx :tllr,ttcd p(‘riod of t imtb. (c) (iet

electrophoresis

Small-scale preparative gels and analytical gels (20 or 25 ctti x 20 cm x 0.3 cm t,ltick) were cast and rtm in a \-ertical apparatus sitnilar to t,lrat described by St)udier (l!l73). Large-scale preparative agarose gel electroptroresis was carried oitt in Irorizontal slab ycls et al., 1974) was used. For agaroac~ (23 cm x 23 cm x 2 cm). In all casts, E httffw (Hcllittp ppl compositions below 2(&, gels were rtttt al I.5 V/cm. l’or rompositiotrs of do{, or Itigltw. t,lte voltage was maintairtcd at 2.5 V/cm. For polyacrylamide gels tttr buffer systjrrtt (PR) of Pracock KSDingham (1968) was r~sotl. Analytical gels (0.1 cm tltick) were run at 8 to 10 V/ctn or (0.3 ctn ttlirk) at, 5 V/cm. For polyacrylamide percent,ages helow~ 5?o. tltcl acrylatnide: his ratio was 20: 1, and W59,, ayarose was added for additional support,. For polyacrylatnide pcrcctttagcs of 5”(, ot greater, the acrylamide: bis ratio \vas 40: 1.3. Gels were stained in cthidium bromitl~~ ( 1 g&l) and visualized witlr a short-\vavcs ultraviolet lamp or stained in a solntjiotr of 50(& fortnamidc, 0.005°& Stains-all ( 1-c$ttyl2.[3-( 1 -ethylnapt,ho[ 1,2d]tttiazolin-2-~lidettr)-2-tnetlt~lpropcrt;yl]t~aptttt~[ 1,2d]tttiazoliuttt bromide). Radioactive gels wcrc fixed for 20 t’o 30 mitt ttt t,hcx fortnamidr/Staitts-all solrttiotr and then autoradiographcd cither at room temperatttw Itsing Kodak NS-54T tttcdical X-ray film, or at - 70°C (Swanstrom B Sttartk, 1978) using Kodak XH-5 medical X-ral film atrtl a Dttpottt (C’rottcx. Ligttttritr g Pltts) or Ilford (l”nst, Ttttrgstat~t~) ittt,csttsif?;ittg scrwtI.

DNA fragments obtaittcxd from agarow were elttted by I of t*lte follouit~p % tnet,hods. Agarose was dissolved itt an equal volume of 8 mII-NaClO,, 0.04 wNaPO, (pH 6.9) at (i5’Y! followed by adsorption to Itydroxylapatitc, (previously degassed and equilibrated bvittt 0.02 nl-NaPO,, pH 6.9). Fragments uwt’ c~ltrttxd from t’ltc, trydroxylapatite Lvitlt 3 to 4 vol. (packed column) of 0.4 M-NaPO,, and tlwn dialyzed c>xtcttsively against 10 tnM-Tris.HCl (pH 7.9). The second, and prr~fwwd. ttwt,ttod for ehtt~iotl of DNA ftxgtn~~nts frotn agarosr is a modification of the Itorizotttal rlectro-clltttiott dtwritx~tl b>- McDonell et al. ( 1977). Ttte gel slices were placed in dialysis bags atttl cllutcd ovrrttigttt at 60 to 70 V using 0.5 x PR buffer. For elation from ncrylamidc gels. gc’l slices \vcr(s pla.cc~l in vertical columns wittt cither sintered glass or tt,vlott tncslt sttpports attd cltctropltorrsed into dialysis tubing attached to t,he outlet, of tttc colttmtt. Elcctro-cltttiott \VAS carricltl ottt itt 0.5 x PR for several hours at 150 V. Eluted fragments were adsorbed to tt small coltttntt of HND-crllttlosc, prcvioust> equilibrated witlt 10 tnpt-Tris. HCl (7.4). 0.3 ~lr-NaCl, 1 tnnl-EDTA. Ttttl colrtmtt \vas washed with several column volrttncs of ttw satnc buffw. Ttrc fragments ~vwt~ eluted witlr at Icast 2 cohimn voltm~w of elntiott bttfl(lr ( I ni-Na(‘l, 1R”,, f~ttlat~ol), precipitated wittr 2 to 3 vol. 95%) ethanol at - 7OV. and washed scvcral t,imrs with !,516 (~tttattol. Recoveries from agarosc and acrylarnide ut,ilizing c,lt.ctro-elutiott ttstially rangrct frotn 50 to SOY/,. The 5’ termini of restriction fragments were labeled wittt [ y-“2P]A’I’P and polyttocleot,itle kinase (Maxam & Gilbert. 1977) as described previously hb- Egatt & Lattcl~~ (1978). Internal labeling (nick translation) of DNA fragment~s was carried out witlt DNA polymerase (Boehringer Mannheim) at, 100 units/ml (K&by et al.. 1977) attd a sit&

01CGASIZATIOS

OF t,RNATYP (:Eh-l?

(‘I,ITS’I’ERS

23

(a-321’)-labeled deoxyribonucleotide triphosphate. Xeactiotls wert’ terminated by 2 extractions with water-sat,urated phenol. Tllis was followed by addit,ion of ammonium acctatr to 2.0 no, and the sample was maintained at room temperature for 20 to 30 min before precipitation with ethanol. Aft,cr labeling by Ilick translation, all fragments VYW Icpllrifirtf I,>- gc.1 elrctrophoresis.

D?CA uxs tratlsferred from agarose gels to nitroccll~dosc filt’ers according to the method of Sollthrrn ( 1975). For hybridizations, filters were rolled and fitted int’o small-diameter t’est tubes containing 3 x SS(’ (1 x SSC’ is 0.15 hI-NaC’l. 0.015 al-sodium citrate), 0.20,, sodiutu dodecyl sulfate, acid ;I I /100 dilut,iotl of tilt* Ilyhridizatio~l lnrdiurn of Denhardt, (1 966). Radioactive DNA fra.grncvit,s it1 10 Ins%-Tris- HCI (pH T-9) were Ileat-denatured ill a boiling-water bat11 prior to Ilybridizatiorrs \vllicll LWW carried otlt at 65°C for 20 II. ll’ilters were nlltoradioaraplleti as described ahovc>. Hybridizatiotl of isolated DNA r(‘ctrictioll fragments with [3H]tKNA% rnricllett for t,lZNA:ATy’ (Landy et al., 1967,1974a) was sarricvl ollt as previously descrihcxd (Landy et al.. 1971n).

‘I’l~t- DNA sec1t~vncirrg procvdrtrc> of Maxatn & (:ilbcJrt (19ii) \vas used witIt tllcx moditlescrihc~d by Egan B Landy (197X). Thcx grl system ([set1 was I;l”& acrylamidv PH elrctrophoresis buffrr. (front n 40:2 acrylatnidc:bix sttrcsk). 8 NI-III’W, \vitll O..i

ficatiorls

T11e restriction fragments wcr(* assigned a ~lamta based 01, tile following factors: (a) tile Z rrst.riet,iori nlzolease sites formirlp t,he ends of tllc fraglncxrit~s: (b) the number of nucleotidc pairs comprising the fragments (det,crrnint~ti from tIltI seqr~rrcc~ or estimated by elpct,rophoretic mobility rrlati\re to known standards) ; ((1) wllrrl tlrcessary, the previously pllblished name of the parent fragmclnt or a Ilame assigned to it, based on it,s mobilit,y relative t,o thrl canonical phage H&II f II I fragmrnts (Lantly et al.. 1974a,c). Additionally, when the parental tllr: satne fragment can be ohtaincd from 2 differerlt phagr,, a suffix describing phage is included, e.g. HaeIILH~inII-400(hr~~d 18) and HaeIIlH?;nII-400(Xt~80dglyTs~~~j) or HinIlHaeIII-185(singlet,) and HinIIHaeITT185(do,uhiet.).

Molrcular wpigtrt estimates for restriction fraglnent)s wvrc determined from their clectrophoret,ic mobility on avrylatnide or agarosc’ gels as compared to a calibratnd standard. ‘For large EcoKl or H&Ill frklgments (Figs 1 alrd !j) ttle markers used were an EcoRI digest, of bacteriophage X DNA (Thomas & Davis. 1!)75), or EcoRI or H&III digests of bacteriophage 480 DNA (James et al., 1976; Moore cY:James. 1978). All other fragment molecular weights were estimated losing eittler Hi,lII + III digests of +8Opsu& (singlet) DNA (Landy et al.. 1974a,c) or Haelll-digested HinIl I- III-73O(Cla-singlet) as molecular weight &mdards. The C to I! group of fragment,s of the HinIl t III 98Opsu&, profile (Landy et al., 1!)74c; also Fip. 5) have been recalibrat’ed against, a collection of 18 fraptnerlts whose exact lengths are now known from DNA sequence analyses. lneludcd ill this group of secluenced fragmrnt,s are 9 fragmcnt,s from t.he bacteriophage X attachmentsite region (Landy & Koss, 1977), 8 fragments from ttltl t,RNATY’ region of 48OpsuA, (singlet’) and 48Opsu:,iunpuh(doubl(,t) (&an dt Land?-, 1978; .J. Egan 8: A. Landy, from the /a,c regulatory region (Landy et al., 19746). Thv lisllcd results), and 1 fragment r(lcali brated values for the HinIl r III +8Ops1;;, fragment,s are indicated in Fig. 5. Ttlcs \-alues for fragments H&II + II I-73O(Cla-singlet). HinIl -+- 111.1020(BlOa-doublet) and H&II1070(38-h1~80dglyTsu~i) wwo derived from the sum of tlteir subfragments, many of which are now completely sequerlcrd (Sekiga et a/.. 1976: Epa.n & Landy, 1978; J. Egan R.A. Landy, llnpublished results). ‘IYI(> suffix .O or .5 (for restriction frapmrtlts \vitil all odd tr\unbcar of unpaired bases ill st,ag:perrd ends) is Ilsed u-lien thcb oxact numt)clr of t)aslL-pairs irl a fragment is ktlown frotrr Wq11etlct~ analysis, c.6. HaeIII-3X.0.

.J . .J . ROSS1 (j) Rnzymex

Restriction

endonucleascs

;Zlul

1’7’ 2

and

(A-GIG-T).

chemicals

~%o)lI

1 HhaI

(U-(‘-G-C).

(referred

HincII

AI d

t’o as HinII)

(GfA-A-T-T-C). I (G-T-P-y-P-u-A-C),

HaeI11 HindLII

(O-c:tC-C), (referred

to

as H&III) (At*-G-U-T-T), Hinfl (GIA-N-T-C), HpaIl (C+-C-c&G) and Pstl (C-T-WA~C:) were purified as described pre\ionsly (Landy et al.. 1974~; Robinson & Landy, 1!177; Egan & Landy, 1978) or pm-chased from New England Biolabs (Beverly, MA). A mixture of Hind11 + III was prepared from the Haewwphilus iwjuenzae &rain Rd (Landy et al., 1974c). For conveniwce. WP refer to t)hr Hin.dII {- TTI activities simply as Hin.11, HinlII, or H&II + III. Polynwleotide killasr was purchased from either P-L Biochernicals. .I no. (Milwaukee, TIN-. (Indianapolis, IN). DNAase I ad bacterial alkaline WI), or Boehringer-Mamllleirn, phosphatase were purchased from Wortbingtolt Riochemicals (Preetwld, NJ). DNA polymerase 1 (E. coli. fraction Wl) was pwcbased from Roehringer-MaIlntleirn, Inc. BND-cellulose (Serva Feinbiochem.) was purebased from Accurate Chrmical and Scientific Corp. (Hicksville, N.Y.). T11e so~~~xx~s of all otlrer ctwmicals and radioactive conpounds lrave bee11 reported prtviously (Landy et al.. 1974~: Hobins(xI & Landy, I977 ; Egan & Landy, 1978).

3. Results (x) IdentQication.

of

DNA fragments

curryiny

the genes for

either

tRNA’fY’ or

tRNATr Linear maps of the four transducing phages used in this study illustrating EcoRI and H&III cleavage sites. as well as the relative positions of the tRNA genes are presented in Figure I. The identification and isolation of Hinll and HinlI + I.Il fragments carrying the tRNAy’ gene from either @3Opsu&, (singlet) or @Opsu& - (doublet) phsges have been described (Landy et aZ., 1974a). The Hi~2Zl + III fragments are depicted in Figure 10 and have the following designations: HinlT + TIT-730 (Cla-singlet) and H&II + III-1020 (BlOa-doublet). A H&II fragment from hhSOdglyTsu& carrying the tRNAy’ gene was identified as follows. Xh80dgZyTsu& and the helper phage hh80 nere digested with HinII + 111 and electrophoresed in 2% acrylamide, 0*5q/l agarose. The transducer unique fragments were eluted from the gel, adsorbed t,o uitrocellulose, and hybridized with a [3H]tRNA preparation enriched for t,RNA Tyr. A HinIl 1070-base-pair fragment was found to hybridize t,o this preparation (data not shown). Confirmation that HinII1070(B8) carries at’ least the gene for tRNAy’ was made by hybridization of this fragment to the tRNAy’ -carrying fragment H&II + III-730(Cla) (singlet) (data not shown). It should be noted that HinIl-1070(B8) has two H&II ends, since the EcoRI 6.5 x 103-base-pair fragment from which it was derived was shown not, to have any

H&III

sites withill

it (see Fig.

1).

The localization of the gene for tRNAp’ on hrifd18 has been described by Nomura (1976). These results demonstrate that this gene is contained on an 8.6 x 103-basepair EcoRI fragment (Fig. 1) and that this fragment is not cut by H&III (Lindahl et al., 1977). By hybridizing a HinII + TII digest of hrifd18 (bound to nitrocellulose (Southern, 1975)) to a #Opsu&, (singlet) HinfI-35.0 fragment, which derives entirely from the mature tRNAy’ gene sequence, we have identified a HinII fragment from a HinII + 111 digest of hrifd18 which carries at least the gene for tRNAp (data, not

ORGANIZATIOS

OF tRNATY’

(:ENE

CLT’STERI’

ECORf &3ops”~Hinm

O$.Gh/?,Tyr2

E?ORI XhsOdglyTsu+,

‘y c(

HinlII

,/*~* (I)

-19.7

’ 9.7

I 1

7.7

1

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EcoR I Xrifd 18 Hinm PI; 103-base-pair AhSOdgZyTsu& t,Rh’AT”‘containing fragment but not t,o the 8.6 x 103-base-pair tRNAy’-bearing fragment from Xrifd18. We have extended t’hese result’s by- hybridizing each of the Heel It tragmcnts derived from upstwam of t,RNAF” on iii/r II +- ITT-73O(Cla) (see Fig. 10) to Hi)l [I-digested @Opsu& ~ hh8OdgZyTsu,‘, and h~if~l8 (data not) shown). ‘I’lrc~ rrsults obtained from each of these experimenth confirmed that, sequences upstream from tR?jA~’ on +30psu1:; ~. ~80psu1~, and t,RKXy’ on hh8Od$yTsu& are homologous for at’ least 500 base-pairs up to the ups’trcam Hi/l11 site on each of the ). Hir/Il {- 1II-730(Cla-480psu~,). ant1 fragment,s Hin I L A- II I -1020( BlOa-#Mpsi& vwy weak. and in some Hi~rl1-107O(B8-hh8OdgZ~~lTsu,+,) (WC Fig. IO). In contrast. eases no. homolog>~ VW sew1 lwt \\ww the tRiYAA~“” upst warn t’ra~rrwr~ts and Hin I I 2700(L43a-h$dl 8). In order to tlctcrminc \vhr+twr or not sc~luw~ws (lo\\ ustrcnm fixm t,hc tRNATY’ and tR,l’Ay“ ywcs were similar, advantapc wa h taken of a pair of 178+-base-pail writs of repeats that begin 19 bastfkymt,nts that. derive centralI>- from an unusual pairs upst,ream from t’he 3’OH end of the tRNAT” mature struc:t,ural sequence (Egan signal KSLandy. 1978) (see Fig. 9). and contain an ir/ vifvo p-dependent termination (Kiippw et al.. 1978). From the results prcwnted in Figure 9. it is apparent’ that there is xwy Mtle homology (besides the 19 base-pairs of matuw tRR’ATY” struct’ural

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I>Io. 10. Final maps of the tRN-4 gene clusters carrirtl 1)~ 4 different transducing phages. and Arqd18. @Opsu,:; - ) Q8Opsu;,,, hh8OdgZyTau,~ Sequences corresponding to (coding for) the mature t,RNAs ( ) and t,ranscriptional tlirH&loll (4) are intlicatetl. The 2 HidTI-178.0 fragments that atljom His11 + III-102O(RIOa) an results). same as that found in the #‘. CYJ/~ chromosome, (unpublished (I)) Th,p matura dructural srque~~us of t RNA1 g”-t RLVA -z’“-t RX;Iy’ are clusterd Pc*ithin approximatdy 350 OCLSP-pc~irs 011XhXOtig1yTsw~6 and hrifdlX The specialized transducing bacteriophages hhXOdgl@u& and Xrifd18 have bot#h been shown to carry genes coding for tRNL4~“-tRNX~1Y-tRNA~hr (Squires et al., 1973: Wu et al., 1973; Daniel et al., 1975: Nomura. 1976). In the experiments report’ed here t,he crit’eria for identification of t,he HilaIr restrict,ion fragments that carry these tR,R;X genes tvere: (a) specific hybridizat,ion of each of the Ili)aLI fragments wit.h ctithrr tRYA?’ or a restriction fragment. carrying tRlVA 7’ information ; (b) demon/A st,ration of restriction fragments t’hat are predicted from t’hc known tRh’A maturcb struc%ural and surromlding sequences: and (c) DXA sequence analysis of DSA fragments carrying either tRNATY’. tRNAzlY. or tRNA, Thr informat,ion. The actfun verification of tRNA7’ on hh8Odgl?/Tsu& (as oppostd t,o tRNAyY’, see helow~) is dependent on the partial fingerprint analyses of Squires Pf al. (1973). 7’htl rlose linkage of the t’RSA~r-tRN~4~‘“-tR~*4~hr gtxncs on hot,h hh8OdgZyil’su,i, and hrifd18 (Fig. 10) is somewhat in agreement’ with the &&on microscopic fcrritinlabcltd tRNA mapping of \%‘11d al. (1973) for Xh8M~/Z@‘su&. Their results mapped the tRNA gem cluster to within a total nequenct~ of approximately 500 base-pairs. Our rcsult,s indicat.e a dist.ance of approximately 350 base-pairs bet,ween the 5’ end of tRKVI-\y’ and the 3’ end oft RNAyr (Fig. IO). Our discwpancy between the present, results and t,hose of Wu et al. (1973) concerns the relat,ivc order of t*he three tRNA ant1 zhr. Our results are yen es. Their data placed tRNATY’ in h&wren tRNXsz’\’ consisttsnt with t)hose of Chang &I Carbon (1975) who isolated a t,RNA~‘Y-tRNA~”

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precursor from E. coli infected with XhWdgZyTsu&. Our results are also consistent with t,he genetic mapping data for t,//rc’ (t,RNATY’) and gZr/T (tRNAzlY) (Orias it rrb.. 1972). (c) The sequewe3 upstrenrtt from tKNATY” itt hhXOdgly’l?su& and Xrifd18 are different, but this region in hhXOdgly’l’su& is hom,ologous to the regicw upstrecrw from tRNATY’ in $XOpsu& (singlet) nn.d @lOpcsu& - (doublr4) One of the most surprising results to come out of the present analyses of the tRNATY’ and tRNATYr2 -tRNAz’Y-tRKAF”-carrying bact)eriophages is the large extent of homology upstream from the tRNATYr genes on the tRNAy’ transducing phages transducing phagc Xh8OdgZyTsu& ( Figs 6. 9 and 10). An identical and tRNATyr pattern of rest’riction endonuclease sites for five different, enzymes. encompassing 13 sites (Fig. lo), cannot be simply fortuitous. ‘I’he results of hybridization of upstream fragments from the tRNATY’ region with Xh8OdgZyTsu& restriction fragment,s contirm this homology (Fig. 9). Based on the sequence analysis of Rekiya rt al. (1976) , some homology was expected out to at least, 59 base-pairs upst’ream from the tjranscription initiation point, but, our results extend t,his homology to over 500 base-pairs (Fig. 10). Since sequences extending only up to the HinIT site 500 base-pairs upstream from tRNATyr have been analyzed. it is conceivable that’ this ho~nolog~ rxt’ends even further upstream. Conversely, the region upst,ream from t’RNATYr on hrijd18 does not share significant homology with the ot,her bacteriophages (Figs 6. 9 and 10). It could be argued that t,he upstream homologies observed between the tRNAFY’ fra.gments and Hinll-1070(B8) are due to @O DNA comn~on t’o these phages, while Xrifd18 cont,ains no $30 DNA. ‘Wis is est~rernrly unlikely. based on a number of different criteria. +SOpsu$ - carries about 34) Y 103 base-pairs of E. coli DNA (approx. 2.7 x lo3 base-pairs in @3Opsu&) locatecl t’o the right’ of the $8Oatt site (Fiandt rt (~1.. 1971 ; Miller ft nl.. 1971 : \V:u & Davidson. 1973) (see Fig. 1). We have eat’ablishcd by restriction enzyme anslyses and hybridization experiments that most of t’hc Ii:. w/i DNA is upst,rram from t,he tRKAy”” gene (sw~ Vip. I). ‘l’he bacteriophage hh8OdgZyTsuj’, carries about 21.2 x 103 base-pairs of E. wli DIVA (W II d ~1.. 1973). and t,hr tRNAzyl’gene clustfar is located vvitjtrin a 6.5 i 103-base-pair EcoRI fragment tRKA;lY-tRNAT’ which maps to the left, end of hh8Odgl,//Tsu,‘, (see Pig. 1). ‘I’he paradox of the dissimilar structures of tJhe regions upstream from tRYA?” on and hrifd18 could rit,her be due to artifacts in the generat’ion of one or hh80dglyTsu& both of these bacteriophages, or it might. reflect the existence of t’wo different, t,RNAy”gene clusters on the h’. coZ,ichromosome. ‘l’he results reported hcrt tRNA;lY-tRh’Ayr make it fca,sible to analyze the E. coli chromosome tlirectjly for the stjructure ant1 organizat’ion of these t,RNA genes : t,he well-charactjerized restriction fragments from different regions of the tRNA genes can be readily used as specific. high-resolution. hybridization probes. Results from studies of this type establish that’ the differences by t,\vo upskeam from tRNATY’ in hhSOdgZyTsu& and Xrrstc,ttt can 1~ ittterpwtetl as indicating differential resistance to rifampicin by the promoter for t RNA?’ and a, l~rtwtmptivt~ protnotw for the tRNA~tY-tR?JA~hP penes (Grimbrrp & 1)anicl. 1977). (‘hang & Carbon (197.5) \vertx utiablt~ to isolate ill eiuo ih precursor containing all thrw tRNAs in an RN.\aw P strain of E. coli harboring X1180t~gzy%;,, wqwticr a precursor containing although tt1c.y tlicl isolat’c and t RNA~‘y-tRK~4~hf.. Ilgen rl ~1. (1976) hare isolat rd a 205base-pair tRNATY” prtatwrsor from att RN-4asr I’- strain of E. roli lysogtwic for hh80d~ZyTsu&, but also did not find a precursor cottt;titting all tttrw tKN.1 pcw~. htr8OdgZyTsu& contains a settc (Srkiva it al., 1976; Kipper km)\\-ti prottiotr~r seqwtiw prwtditig t8ht~tRNAr\” c~f/I/.. 1975). t trus making it tiwible that tratwriptiott for a tricistrottic precursor wultl initiate t1we. ;\t prtwstit. little is known conwrtiing the regulatory or coding wgic:tts itp~trratii from t,RSAT”” on hrifdl 8 or t It-t)alski, \V. (I!IiJ). .I. .Ilo/. fjiol. 56, X3- 368. Moorf~. S. K. & .lames, E. (1978). (:wre. 3, 53 80. Norllllra, hf. (I!4S6). Cell, 9, 633 ti44. Orias. H.. (:arttifsr. ‘r. K., Lttnnan. .I. E. di Bdach, M. (19i2). .I. Uacten’ol. 109, 1125 -1 133. Pww wk. A. C’. & Dingllarn. (‘. \\‘. (1!)68). Biochemistry. 7, 668 674. J-Zigt)j,, T’., Dir~kmarm, M., Jil~~t-lt~s, C’. & Bclrg. I’. (l!l77). ./. LZlol. I?io/. 113, 2X7 251. Kobinson, L. H. K: Lancly. A\. ( I !17i). (he. 2. I ~3 I. lir~src~ll. K. L., Ahlson. J. N.. IAII~~, A.. (W’trr. M. I,.. Ilrcatltlrr. S. & Srnitll. .I. 1). (1970). .I. Jlol. Hiol. 47, 1 13. S~~lrcdl. t’.. lGrt)c:rts, .I. & Prirnakoff. I’. (llf76). Cell. 8. 581 5!)4. SvkiJ-a. ‘I’. Hr. Khorana, H. (G. (1974). l’roc. Sat. Acad. h’ci.. I’.S..4. 71, 2978-2982. Rvki>,a. ‘I’.. XXII Ormontlt, H. B Kllorana, H. G. (1075). .I. Bid. Cherra. 250, 1087-1098. H. Ct. (1976). .J. Biol. Chem. Seki>-it. ‘I’.. Cotltwras, H.. Kiippcr. H.. Larrtl> . A. & Klloratra. 251, 512-C 5140. SigrltLr, E. I

Structural organization of Escherichia coli tRNAtyr gene clusters in four different transducing bacteriophages.

J. Nol. Bid. (1979) 128, 2147 Structural Organization of Escherichia coli tRNATYr Gene Clusters in Four Different Transducing Bacteriophages tI~~-~...
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