Plasmid R6K DNA replication

.I. Mol. Biol.

(1982)

161, 33-43

Plasmid R6K DNA

Replication

I. Complete Nucleotide Sequence of an Autonomously Dsv111 M. STALKER, ROBERTO

KOLTERt

Replicating Segment

AND DONAM) R. HELINSKI

Department of Biology University of Calijornia, San Diego La Jolla, CA 92093, U.S.A. (Received

11 June 1981, and in revised form 9 June 1982)

A 1565.base segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichin coli. This segment contains a functional origin of replication and the structural gene pir for a protein, designated n, that is required for the initiation of R6K DNA replication. The nucleotide sequence of this 1565 base-pair replicon was determined. From the sequence and the analysis of proteins produced by minicells of pir gene and a deletion of the pir gene, it can be E. coli strains carrying the wild-type concluded that the n structural gene encodes for a protein of a molecular weight of approximately 35,ooO. On the basis of the nucleotide sequence, the n protein is the only protein containing more than 50 amino acid residues that is encoded by this 1583 base-pair replicon. The nucleotide sequence also contains eight 22 base-pair direct repeats. Seven of the direct repeats are in tandem and located in the origin region, while the eighth repeat is near or part of the promoter for the n structural gene. This eighth repeat may play a role in the autoregulated expression of the r structural gene.

1. Introduction R6K is a self-transmissible, multicopy plasmid that is 38 kb$ in size and specifies resistance to the antibiotics ampicillin and streptomycin (Kontomichalou et al., 1970). A physical and genetic map of plasmid R6K had been constructed that included two bidirectional origins of DNA replication in Escherichia eoli, designated N and /3 (Lovett et al., 1975; Crosa et al., 1976), and an asymmetric terminus of replication (Lovett et al., 1975; Kolter & Helinski, 197&x). However, a recent analysis of replicative intermediates of RSF1040, a spontaneous deletion mutant of R6K synthesized in vitro, revealed initiation of DNA replication from three distinct origins (Inuzuka et al., 1980). Two of the origins of replication in vitro correspond to the origins N and fl identified in vivo, and the third to a new origin termed y, which is located between cxand B (see Fig. 1). A more detailed analysis of the utilization of the a and p origins in vivo revealed that a portion of RSF1040 molecules also initiate at the y origin (Crosa, 1980). 7 Present address: $ Abbreviations

Department of Biology, Stanford used: kb, lo3 bases; bp. base-pair.

University,

Stanford.

CA 94305,

U.S.A

33 0022%2836/82/290033-11

$03.00/O

0

1982 Academic

Press Inc.

(London)

Ltd.

34

D. &I. STALKER,

R. KOLTER

AND

D. R. HELINBKI

Construction of low molecular weight derivatives of plasmid R6K has defined a 25 kb segment of the plasmid that contains sufficient information for autonomous replication in E. coli (Kolter & Helinski, 19786). This segment contains the y origin of DNA replication and a structural gene (pir) that codes for a protein (designated r) that is required for the initiation of R6K DNA replication in vitro (Inuzuka & Helinski, 1978). The pir region is located in two adjacent Hind111 fragments (designated 9 and 15) in the 25 kb segment of R6K (see Fig. 1). The y origin of replication is located in the region of the junction of Hind111 fragments 4 and 9. Hind111 fragments 9 and 15 do not in themselves constitute a functional replicon. However, when these two fragments are joined to a ColEl replicon or a lysogenized phage lambda derivative, they will support in trans the replication of y origin-containing derivatives of R6K (Kolter et al., 1978). These two fragments, however, will not rescue in bans plasmid derivatives of R6K containing either the LY or /3 origins without the y origin region (Shafferman et al., 1981). The nucleotide sequence of a 520 bp portion of R6K containing the y origin of replication has been described (Stalker et al.. 1979). In this paper, we present the nucleotide sequence from the y origin region through the structural gene for the n protein, which is adjacent to the y origin. The entire 1583 bp sequence encompasses all of Hind111 fragments 9 and 15, a 200 bp portion of Hind111 fragment 2, and 110 bp of Hind111 fragment 4. This nucleotide sequence is capable of functioning as an autonomous replicon in E. coli (Kolter et al., 1978). In addition, on the basis of the analysis of proteins produced by minicells of E. coli carrying the wild-type pir gene and a deletion of the pir gene that specifies a truncated rr protein, direct evidence is provided for the pir gene as the structural gene for the n protein.

2. Materials (a) Enzqlmes

and Methods (I nd isotopes

The restriction endonucleases HindHI, HueII. HaeIII and BgZII were purified by the procedure of Green et al. (1978). TaqI, AZuI and H&f1 were obtained from New England Biolabs. Bacterial alkaline phosphatase and phage T4 polynucleotide kinase were from respectively. The large fragment of E. coli DNA Worthington and P-L Biochemicals, polymerase I was purchased from Boehringer-Mannheim. The Radiochemical Centre was the source of [Y-~‘P]ATP (3000 Ci/mmol). [‘t-32P]dNTPs (350 Ci/mmol) and L-[~~S] methionine (800 to 1100 Ci/mol). (b) Plasrnids

arid strains

Plasmids R6K, pRK353 and pRK419 were maintained in E. coli strain C600. Strains carrying these plasmids were grown in L-broth and the covalently closed circular DNA forms purified from Sarkosyl lysates (Bazaral & Helinski, 1968) by 2 centrifugations in CsCl/ethidium bromide gradients. The plasmid DNAs were precipitated with ethanol and dissolved in the appropriate buffers for digestion with restriction enzymes. E. coli strain YSl was used for mini-cell preparations (Inuzuka & Helinski, 1978). (c) Labeling

and purijkation

of DNA fragmerds

Approximately 1 to 5 pg of plasmid DNA were digested with the appropriate restriction endonuclease and the 5’ ends labeled by the polynucleotide kinase reaction with [Y-~~P]ATP (Murray, 1973). DNA fragments containing 3’.recessed ends were filled in usin the large f (a-3 P)-labeled fragment of DNA polymerase I (Klenow et al.. 1971) and the appropriate

PLASMID

R6K

DNA

REPLICATION

I

35

deoxyribonucleotides. DNA fragments to be sequenced were purified on 6% (w/v) polyacrylamide gels and eluted with a buffer containing 05 M-ammonium acetate, 0.01 Mmagnesium acetate, 001 M-EDTA and 91% (w / v ) sod ium dodecyl sulfate. DNA fragments labeled at both ends were cleaved with another restriction enzyme or subjected to strand separation on 5% polyacrylamide gels (Maxam & Gilbert, 1977). Labeled fragments were eluted as described above and precipitated in the presence of excess transfer RNA and dried. (d) DNA sequence determination All chemical degradations were carried out according to Maxam & Gilbert (1977) with modifications described elsewhere (Stalker et al., 1979). A sequence of up to 250 bases could be obtained readily from a respective end-label. (e) Minicell preparations Minicells were purified as described (Meagher et al., 1977). Minicells obtained from a 509ml culture were washed and resuspended in 5 ml of assay medium containing M9 salts, 95% (w/v) glucose, 25 pg thiamine/ml and 2Opg of an amino acid mixture (except methionine)/ml. After incubation for 20 min at 37°C and labeling for 20 min at 37°C with 30 &i of L-[35S]methionine, the minicells were pelleted at 10,OOOgfor 5 min and frozen. The pellets were resuspended in 504 of sample buffer (Laemmli, 1970) and heated at 90°C for 5 min. Samples (10 ~1) were electrophoresed on a sodium dodecyl sulfate/l2.5:/, polyacrylamide gel (Laemmli, 1970). The gel was stained with Coomassie brilliant blue, destained and dried, and the labeled proteins identified by autoradiography.

3. Results (a) Determination

of the nucleotide

sequence

As shown in Figure 1, the nucleotide sequence that was determined included Hind111 fragments 9 and 15 in their entirety, a 110 bp segment of Hind111 fragment 4, and a 200 bp portion of Hind111 fragment 2. Hind111 fragments 4 and 2 lie adjacent to the ends of fragments 9 and 15, respectively, on the R6K map. A single DNA preparation of a small autonomously replicating R6K derivative, pRK419 (Kolter & Helinski, 197&z), was used to determine the total sequence containing the pir gene region, while a single DNA preparation of pRK526, a plasmid derived from pRK419 and containing the y origin of replication, was employed for the nucleotide sequence of the y origin region previously described (Stalker et al., 1979). The strategy for sequencing the R6K replication region is shown in Figure 2. Both the 3’ and 5’ ends of the HindIII, BgEII and Tag1 restriction endonuclease sites were labeled and the sequence determined in each direction from these sites for both complementary DNA strands. The small H&f1 restriction fragments were isolated from HinfI digests of pRK526, labeled at their 5’ ends, strand separated and sequenced. This overall strategy facilitated significant overlap between sequences obtained from individual restriction fragments and made possible determination of each nucleotide position in both complementary DNA strands. (b) Significant

features

of the nucleotide

sequence

One strand of the total 1565 bp sequence of the minimal R6K replicon is displayed in Figure 3. The entire sequence is numbered in both directions, starting from the Hind111 site located at the junction of Hind111 fragments 4 and 9. The

D.

36: 4

M.

STALKER,

H/ndBI

Bg/lI

R.

KOLTER

9 II

II

AND

D.

BgfII HIndIll

K. 15

HELINSKI 2

HmdJIl

T&l

,

, ’ ’

FIG. 1. A physical map of the antibiotic resistance plasmid R6K is shown. 1, p and y refer to the 3 origins of DNA replication. ter refers to the asymmetric terminus of replication. The arrow indicates the single BamHI site located in the ampicillin resistance gene. Hind111 fragments in the R6K replication region are designated 4, 9, 15 and 2. The region that was sequenced is expanded. Relevant restriction endonuclease sites are indicated. HaeII sites are designated II. pRK353 and pRK419 are self-replicating derivatives of plasmid R6K.

sequence is 62% rich in adenine and thymine base-pairs, which is somewhat higher than the sequences obtained for the replication regions of ColEl (Oka et al., 1979), phage lambda (Grosschedl & Hobom, 1979), and RlOO (Rosen et al., 1979). The most striking feature of the nucleotide sequence is the presence of eight 22 bp direct repeats, which are indicated by arrows in Figure 3. Seven of these direct repeat units are in tandem and span base positions 72 through 241, which is a region essential for functional y origin activity. The minimal sequence required for a functional y origin has been described (Kolter et al., 1978; Stalker et al., 1979) and includes a region from - 100 bp to a BgZII restriction enzyme site located at the

Hh0I

t HueIII

t HoelI

t

t

HaeII

HP01

FIG. 2. The strategy of DNA sequence determination is shown. sequences obtained by 5’.end labeling, while broken arrows refer Restriction endonuclease sites for the entire region are indicated.

Unbroken arrows refer to DNA to sequences from 3’-end labels.

PLAHMID

RBK

DNA

REPLICATION

3i

1

TGTCAGCCGTTAAGTGTTCCTGTGTCACTGAAAATTGCTTTGAGAGGCTCT~AGGGCTTCTCAGTGCGTTA~ATCCCTGGCTTGTTGTCCA -80 -50 -100 -40 -20 .

~CTTTAAAAGCCTTATATATTCTTTTTTTTCTTATAAAACGAGCTTAGTA 40 60 20

80

100

. ~A~CATGAGAGCTTAGTACGTTAG~~ATGAGAG~TTAGTA~GTTAG~~ATGAGGGTTTAGTT~GTTAAA~ATGAGAGCTTAGTACGTT~CAT Ii0 160 Ii0 Ii0 2dO zio

l . ~AAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAACTG~TGAT~TTCAGAT~TAG~TTAAAA~AGGTGGCTTTTTAATCATCTTTGCC nia 26’0 2io 3&l 3iO

.

.

TTGGGGTAATATAGCGACTCATAAAAGCGTTAAACATGAGTGGATAGTAC~~~AGATA~AATTGA~TATTGGCGTT~GATATACAG~T rdo uio r;o 380 nao Go Met-lip

-Leu-Ly8-VaE-M~t-Met-As

-VaZ-ABn-LyS-LyS-Thr-Lys-lte-llrg-Xis-lr

~J~EE~$TTTTT ATGAGI CTCAAGGTCATGATGGAGGTGAACAAAAAAACGAAAATT CGCCACCGAAACGAGCTAAATCACACC rio zob 52b *5b ~~u-ata-Cln-~eu-Pro-Leu-Pro-ALo-Lys-Arg-"~z-~~~-Ty~-~~~-~z~-~~~-~z~-~~~-~z~-*~p-~~~-~~~-~l~-~~~-~~~-~z~-~~y-~zy

CTGGCTCAACTT CCTTTGCCCGCAAAGCGAGTGATGTAT ATGGCGCTTGCTCCCATT GATAGCAAAGAACCTCTT GAACGAGGG ado 660 sdo Go ars-“at.Phe-~ys.~Ze.nrg-Ala-Ctu-Asp-Leu-~z~-*~~-~~~-~z~.‘y*-~z~-Th*-~~~-~~~-~~~-~z~-Ty~-~~y-~z~-~~~-~y~-~z~-~ly CGAGTTTTC AAAATT AGGGCTGAAGACCTT GCAGCGCTCGCCAAAATCACCCCATCGCTTGCTTAT CGACAATTA AAAGAGGGT 6iO 6 6.0 640 6iO Gl -!5ys-Leu-Leu-GZ

-AZo-Ser.LyS-IZe-Ser-Leu-Arg-G(y-As

-Asp-lle-lza-Aza

leu-Ala

lys-Ctu-~~u-nsn-~eu-Pro-Phe-Thr

GGf AAATTA CTT GG’iGCCAGCAAAATT TCGCTAAGAGGGGA?GATATCATT GCT-TTAGCTAAAGAGCTTAACCTGCCCTTT ACT 750 Ii0 7io 7Db AZo‘-‘ s-*sn-Ser.Pro-Glu-GZu-Leu-As -Leu-A8n-ize-IZe-CZu-Trp-12e-AZa-T F-Ser-Asn-As -c*u-Gl -T r-.Leu-ser.Clu-L B GCTAiA AACTCCCCTGAAGAGTTA GA?CTTAACATT ATT GAGTGGATA GCTTiiT TCAAAT GA?GAAGG#Tk TTGTCT TTA AiA 60 BOO aio s*b sin Phe-Thr-Ar

-Phr-Ile-Glu-Pro-T

r-lte-Ser.Ser-G2u.lte-CL

-L

B-L

s-lsn-L

s-Phe-ThP-ThF-Cln-Ctu-Gtu-Thr-llo-Ssr-Ct~

TTC ACCAGi ACCATA GAACCAT&T ATCTCTAGCCTT ATT GG! AiA A8A AATAjjA TTC ACAACGCAATTGTTA ACGGCAAGCTTA ma GO 960 920 Arg-Leu-SeF-Ser-GZn-Tyr-Ser-Ser-Ser-Leu-Ty~-~Z~.~~~-~Z~-~~~-~ s-His-T F-Ser-Asn-Phe-6 s-L a-Lys-Am-T F-Phe-lte CGCTTA AGTAGCCAGTAT TCATCT TCT CTTTAT CAACTTATCAGGAiiG CATTiC TCT AAT TTT AiG AiG AAAAATTiT TTT ATT lOi0 9bo 48.” Id00 I*s-se~-“al-ns -GZu-‘eu-L *-Czu-Ctu-~eu-~te-nzo-T r-Thr-Phe-As -6 S-AS -Cl -Isn-Ile-Cl”-T F-L 8-T F-PFO4B -me ATT TCCGTTGA?GAGTTA A#GGAAGAGTTA ACAGCI TiT ACTTTT GAPAiA GA?GGi AATATT GAGTk AiA Tk CCTGAf TTT l&O 1160 lOi0 loeb Pm-i’te-Phe-L

8.~7

-AS

-“ai-ku-~sn-~

8-lto-1le-~2n-~lu-1le-~

S-L

S-L

8-~hr-~lu-1~e-Ser-Phe-Vat-Gt

-Phe-Thr-Vat

CCTATT TTT AiiA AG&GA?GTGTTA AAT Ai(A GCCATT GCTGAAATT A#A AiG AiA ACAGAAATA TCGTTT GTTGGijTTCACTGTT lii0 11bo 11ro 11’60 “is-GZu-L

a-Glu-Cty-lrg-L

B~lle-Ser-‘ys-LeU-Ly8-Phe-Ctu-Phe-Vot-Vot-~~

-Ctu-As

-CZu-Phe-Ser-Ct

-A8

-Ly8-la

-A#

CATGAAAiA GAAGGAAGAA#A ATT AGTAAGCTGAAGTTC GAATTT GTCGTTGA?GAAGAPGAATTT TCT GG?GAPAAAGA?GAP 1nao 12bo 12io ,260 lib0 Glu-Ato-Phe-Phe-Met-Asn-Leu

Sep

cLu-ata-ns

-A&-,,Za-~he-~eu-~

s-“at-the-as

-c~u-T~F-Y=~~-PFo-PF~-~

GAAGCTTTT TTT ATGAAT TTA TCT GAAGCTGA?GCAGCTTTT CTC.AiG GTATTT AA? GAAACCGTACCTCCC& 1ebo 13io Ii20

8-L

8-Ata-L

8

AiiA GCTAiG 13QQ

6% ?:A” TATATGGCTAAAATTTACGATTTCCCTCAAGGAGCCGAACGCCGCAGGATGCACCGCAAAATCCAGTGGAACAACGCTGTAAAA lp’80 L4’00 I.‘20 1uia FIG. 3. The entire nucleotide sequence of the y origin and the pir gene region is presented. Bases arc numbered from the Hind111 sites starting at position 1. Arrows indicate the positions of the eight 22 bp direct repeats. Boxes outlining DNA sequences refer to RNA polymerase recognition and hinding sequences and a rihosome binding sequence. Amino acids of the n protein are shown above t.hrir respective oodon triplets. Sequence hyphens have been omitted for claritv.

38

I). M. STALKER.

K. KOLTER

A?iD

I). R. HELINSKI

276 bp position. Further delineation of this region and the actual sequences required for y origin activity is presented in an accompanying paper (Kolter $ Helinski, 1982). The eighth direct repeat, located approximately 120 hp from the end of the seven tandem repeats. is not required for a functional y origin. Direct repeats of a nucleotide sequence have been identified in the replication regions of plasmid RK2 (Stalker et al., 1981), mini-F (Tolun & Helinski, 1981), phage lambda (Grosschedl &, Hobom, 1979) and the oric’ regions of E. coli and Salmonella typhimurium (Zyskind & Smith, 1980). Another interesting feature of the y origin region is the 80% A +T rich stretch from base-pairs 1 through 100. Runs of adenine and thymine base-pairs are common to a variet’y of replication origins (Grosschedl & Hobom, 1979; Oka et al., 1979; Stalker et al.. 1981). The putative promoter sequence for the pir gene is located in the region of the eighth direct’ repeat unit The sequence T-CT-C-A-T-G at positions 409 through 415 is homologous to the sequence T-A-T-N-AT-G, which was originally described by Pribnow as an RNA polymerase binding sequence for promoters of the T7 phage (Pribnow, 1975). A 12 bp sequence similar to the RNA polymerase recognition sequence that has been identified for the promoter regions of many prokaryotic structural genes (Rosenberg &, Court, 1979) is located 16 bases upstream. The TTG bases in this sequence are t,he most highly conserved bases of these known RNA polymerase recognition sequences. It is also interesting that the TTG triplet in the 12 bp sequence overlaps the end of the eighth 22 bp repeat unit. The results of transcribing purified DNA fragments from this region are consistent with a major transcript initiating at or near position 421 (R. Kolter, unpublished results). At, base positions 442 to 448 is the sequence A-T-G-MX~-T, of which six of seven bases are complementary to the end of the 16 S ribosomal RNA that is involved in ribosome binding (Shine & Dalgarno. 1974). An open reading frame for the 305 amino acid protein starts (ATG codon) from position 455. There are three recognition sites for the restriction endonuclease HinfI (G-A-N-T-C) located at positions 346, 406 and 459. These enzyme cleavage sites facilitated physical separation of Rr\‘A polymerase binding, ribosome binding and initiation coding sequences from the n structural gene region (Shafferman et al., 1981). The P-origin of R6K Dh’A replication, mapped by electron microscopic analysis of replicative intermediates synthesized in vitro (Inuzuka et al., 1980), is located in a region near the Hind111 junction of fragments 15 and 2 (base position 1284). If the p-origin lies in this region. it shares little detectable homology with the y origin sequence. An interesting sequence in t’his region is an 18 bp palindrome containing all purine residues (position 1204 through 1220). The segment is strikingly similar. hut not exactly homologous to a similar purine-rich region found in the lambda to a origin of replicat’ion (Grosschedl oi Hobom. 1979), and is also complementary pyrimidine stret,ch located in the y origin sequence (positions 17 to 36).

(c) Proteins

coded for by the pir yenr sequence

A likely sequence coding for the 71protein begins with an ATG initiation codon at position 455 and terminates with a TGA ochre codon at position 1370. This rr protein sequence is 305 amino acid residues in length, generating a protein of

PLASMID

R6K 9

e

REPLICATION

I K

-D

I

I

t

DN4

,

“,

’

, <

’

,

1

, ( , (”

/// / I II I 1 I I I, II

I

’

I III

(,

/

/

II I

/

I

,

,

,I,,, I

1

M

/ I ,,I

/

III,

/ I”“11

I

//

I, I I I

I,

I I

1

FIN:. 4. Reading frames for translation of the R6K replicon sequence are shown. Ori refers to the y origin region and P, is the promoter for the pir structural gene. The arrow indicates the dire&m of transcription. Below the restriction map are the 6 possible reading frames for translation. Hash marks above the lines indicate STG initiation starts. while hash marks below the lines indicate TGA, TAA and TAG terminat,ion radons. The filled bar refers to the large open reading frame for the n protein.

approximately 35,000 Mr. The protein sequence contains 339, charged amino acid residues: 38 lysine, 14 arginine and 4 histidine are the positively charged residues, while 30 glutamic acid and 19 aspartic acid make up the negatively charged residues. The protein thus has a net positive charge, which may be important in its role in the initiation of DNA replication and possible association with the y origin region. The protein sequence is unusual, in that it’ contains only one tryptophan residue and no cysteine residue. Approximately 50% of the protein sequence contains amino acid residues having aliphatic side-chains. As shown in Figure 4, the 7r protein is the only major protein coded for by the entire nucleotide sequence displayed in Figure 3. All three reading frames of both complementary DNA strands are closed in both directions by termination codons. Other than V, the largest hypothetical polypeptide found in the pir region is 47 amino acid residues in length (base positions 439 through 583), in the direction of v protein transcription but in a different translation reading frame. The region encoding this hypothetical protein has no DNA sequence resembling ribosome binding sites.

(a)

Km ___

Ib)

(c)

(d)

-

IQ<:. 5. Analj-sis of preparations of h’. roli minicclls cwrlt,itining plasmid RBK and its derivatives. Lanes (a) through (d) indicate results with 4 minicell-producing strains containing plasmids pMK20, pRK419. RGK rind pRK353. respt:c~tivrl\-. The IJNA preparations wcw run on sodium dodwyl sulfate/polgacrglamide slal) gels. pMK20 is il t!olEl derivative t.ha.t spwifiw kanamycin resistance. w refers t.o the position of thta wild-type T protein. 35.000 ,Wr. r* refws to the 32.000 A’, truncated n polgpeptide. Km refers to the protein encoded I)?; thv kanamycin resiutanw grnr.

(d) Evidence

that the pir gene codes for a 35,000 M, protein

in vivo

During the construction of self-replicating low molecular weight derivatives of plasmid R6K, it was observed that Hind111 fragments 9 and 15 in themselves would complement in tram low molecular weight derivatives of R6K containing the y origin region (Kolter et al.. 1978). The putative amino acid sequence of t,hr x protein displayed in Figure 3 indicates that 30 (:-terminal amino acid residues ot the prot,ein reside outside Hind111 fragments 9 and 15 and in Hind111 fragment 2. If 7~protein is synthesized from this region in Gvo, then self-replication derivatives of plasmid R6K missing HindIT fragment 2 should produce a truncated 7~protein. This was tested by preparing minicells containing plasmid R6K and two of its autonomously replicating derivatives and radioactive labeling of the proteins synthesized in these minicells. Lanes (c) and (b) of Figure 5 represent the labeled proteins synthesized in minicells carrying plasmid R6K and pRK419. respectively. Roth plasmids contain intact pir structural gene regions and produce prot’eins wit’h molecular weights of 35,000. Minicells containing plasmid pRK353 (lane (d)), which cont’ains Hind111 fragments 4. 9 and 15 and lacks Hind111 fragment 2. produce a prot,ein of approximately 32,000 L%!~and no protein of 35.000 B’,. These results. which are consistent with the nucleotide seyuencr information, indicate that, the sir structural gene region produce a 35.000 M, polypeptide. and that this protein funct~ions in trans in the replication of y origin-containing derivatives.

4. Discussion We have determined a 1565 bp sequence from t,he antibiotic resistance plasmid R6K, which specifies sufficient information for autonomous replication in E. coli. This sequence contains the y origin of DNA replication and the coding sequence for a protein (n) that is required for initiation of R6K DNA replication (Inuzuka &r Helinski. 1978). On t’he basis of possible open reading frames. the n protein is the only major protein encoded by this autonomously replicating segment. The only other hypothetical polypeptide encoded by this segment is 47 amino acid residues in length but its nucleotide sequence is not preceded by a DNA sequence resembling a ribosome binding sit,e. Consequently, consideration of a role for a plasmid specific protein in the regulation of the copy number of this replicating segment must be confined to the rr protein. The TTprotein is a basic polypeptidc 305 amino acid residues in length that will act in trnns to rescue in ~ivo derivatives of R6K cont)aining the y origin (Kolter f,f 111.. 197X). Sequences containing the 7~ protein, however, will not, t)ranscomplement derivatives of RAK containing either t)he n or /3 origins, despite the requirement of this protein for initiat,ion of replication from either of these two origin sequences (Inuzuka et al., 1980). This is consistent with recent studies indicating a requirement in cis for a functional y origin region for the activity of the or and /3 origins of R6K (A. Shafferman & I). R. Helinski. unpublished results). Recent evidence indicates that t,he r protein both recognizes DNA sequences in the y origin region containing the seven 22 bp direct repeats, and interacts with a region c~onbaining the eighth direct repeat located near the promoter for the n

I). M. S’PALKEK.

41

It. KOLTER

AND 1). R. HELINSKI

structural gene (M. Inuzuka, unpublished results). These observations, coupled with the elucidation of the nucleotide sequence of the y replicon, suggest that n may function at the y origin in the initiation of R6K DNA replication and also interact with the rr promoter sequence to regulate its own expression. Autoregulation of expression of the n structural gene, the possible regulatory role of the n protein for the initiation of R6K DSA replication and the delineation of the R6K y origin of replication are subjects of the accompanying papers (Kolter & Helinski. 1982: Shafferman rt ul.. 1982). The authors thank d. Shafferman for critical reading of the manuscript and helpful discussions. This work was supported 11~grants from the National Institutes of Allergy and Infectious Diseases (AI-07194) and National Science Foundation (l’CM77-06533). One author (D.JI.S.) was supported 1)~ a I-nited States Public Health Servire postdoctoral fellowship (1 -F32GM06542).

KFFFRFW'FS A A

IA

1)

Bazaral. M. & Helinski, D. R. (1968). J. Afol. Kio2. 36, 185194. Crosa. J. H. (1980). ./. Riol. C’hew~.255. 11067-11070. Cross. .J. H.. Lut’tropp. L. K. & Falkw. S. (1976). ./. Bacterial. 126, 454-466. (kreen. P. ,J.. Heyneker. H. L.. Rolivar. F.. Rodriguez. R. I,.. Betlack. M. C’.. Corarubias. -4. A.. Rackman. Ii.. Russel. 1). *J.. Tait. R. Hr Koyer. H. IV. (1978). Sucl. .-1cids Res. 5. 2373-2380. (:rossehedl. R. & Hohom. (+. (1979). ,X’uturv (/,o~do~), 227. 621-627. Inuzuka. M. 8z Helinski, D. R. (197X). Proc. AY~lt..-Ic&. Sci.. 1’9.A. 75, 5381b5385. Jnuzuka. S., Inuzuka. M, & Helinski. I). R. (1980). .J. Biol. Chcm. 255. 11071Pl 1074. KlenoLv. H.. Overgaard-Hansen, K. & Patkarr. S. A%,(1971). Ew. ./. Biochenz. 22, 371-381. Kolter. R. & Helinski. D R. (1978a). ./. Mol. KS. 124. 428 441. Kolter. R. & Helinski. D. R. (197%). I’lusmid. 1. 57lL.589. Kolter. R,. & Helinski. 1). R. (1982). ,I. Mol. Biol. 161. 4.5&.56. Kolter. R.. Inuzuka. M. B Helinski. I). R. (1978). (‘e/l. 15, 119991208. Kontomichalou. P.. IVIitani. XJ. $ Clowcs. R. (‘. (1970). ./. Brcctuiol. 104. 34-43. Laemmli. V. K. (1970). ,Vrrturr (Londorr). 227. 680-685. Lorett. M. A.. Sparks. R. K. B H eIinski. I). R. (1975). I’roc. Sf~t. Acad. Sci., lT.S.,1. 72, 29052909.

Jlaxam, A. B ($ilbcrt. W. (1977). I ‘rot. .w. .-icod. Ax.. l’.S.d 74. 560-.564. JJeagher. R. I~.. Tait. R. C’.. Betlack. M. 6 Royer. H. &‘. (1977). Pell. 10, 52-536. JIurray. K. (1973). Hiochem. .J. 131. 569-576. Oka. A.. Yomura. N.. &Jorita, M.. Sugisaki. H.. Sugimoto, K. & Takanami. M. (1979). Mol. (:rn. (:rnet. 172. lr,lbI:iR. l’ribnow. D. (1975). I’roc. Sat. Acud. ~Sci., i’.S.d. 72. 7844789. Rosen. ,I.. Ohtsubo. H. & Ohtsuho. E. (1979). Mol. Gen. (:enrt. 171. 287.-293. Rosenberg, M. 8r (‘ourt. I). (1979). =I II,,!,. Rec. C:enrt. 13. 319-354. Shafferman. il., Stalker. I). M.. Tolun. A.. Kolter. It. & Helinski, D. R. (1981). In Molecular Biology. Pathogrnicitg axd Ecology of Hacterinl Plrrsmids. pp. 259-270, Plenum Press. Xew \ ork. Shafferman, A.. Kolter. R., Stalker. D. & Helinski, 1). R. (1982). J. Mol. Biol. 161. 57-76. Shine, J. S: Dalgarno. I,. (1974). I’roc. .Vnt. rlcad. SC;.. T-S.,3 71, 1342-1346. Stalker. I). 31.. Kolter. R. 8r Helinski. D. R. (IBit)). Prop. AVat. =Icad. Sci., F.A’.A. 76. 11501154.

PLARMID

R6K

DSA

REPLICATION

I

Stalker, I). M.. Thomas, C. & Helinski, D. R. (1981). Mol. G’rn. Grnet. 181, 8-13. Tolun, A. & Helinski, D. R. (1981). Cdl, 24. 687-694. Zyskind, .J. & Smith, D. W. (1980). Proc. ,Vat. ilcad. Sci., IT.S.d. 77, 2460-2464.

Edited by hf. Gottesman

43