DNA AND PROTEIN INTERACTIONS IN THE REGULATION OF … · DNA AND PROTEIN INTERACTIONS IN THE...

y. Cell Sci. Suppl. 7, 15-31 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987

15

D N A A N D PROTEIN I N T E R A C T IO N S IN TH E

R E G U L A T I O N OF PLASMID REPLIC ATIO N

M A R C I N F I L U T O W I C Z , M I C H A E L J. M c E A C H E R N , P R A D I P

M U K H O P A D H Y A Y * , A L A N G R E E N E R , S H E N G L I Y A N G f a n d

D O N A L D R. H E L I N S K I

Department o f Biology, B-022, University of California, San Diego, La Jolla, California 92093, USA* Department o f Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA■fShanghai Institute o f Materia Medica, Chinese Academy o f Sciences, 319 Yue-Yang Road, Shanghai 200031, China

S U M M A R Y

As for bacterial and animal viruses that employ different mechanisms for their duplication in a

host cell, plasmids have evolved different strategies to assure their hereditary stability or

maintenance at a specific copy number during cell growth and division. A characteristic feature of

plasmid replication control, however, is an involvement of one or more negatively controlling

elements. Furthermore, a majority of the bacterial plasmids examined to date contain direct

nucleotide sequence repeats at their origin of replication and encode a replication protein that binds

to these repeat sequences. The binding of the replication protein (n protein) specified by the

antibiotic resistance plasmid R6K to a set of 22 base pair direct nucleotide sequence repeats is

required for the initiation of replication at each of three origins of replication (a , ¡3 and y) within a

4 K b segment of R6K. The n initiation protein is multifunctional in that it has both positive and

negative activities in both controlling the initiation of replication and autoregulating its own

synthesis. Similarly, the direct repeats of plasmid R6K and several other plasmid systems play

more than one role in plasmid replication. These repeats, termed iterons, are not only required for

origin activity but also exert a negative effect on plasmid copy number possibly as a result of their

‘titration’ of a plasmid encoded replication protein. The properties of plasmid replication proteins

and direct nucleotide sequence repeats that are important for their opposing positive and negative

roles in the regulation of the initiation of replication are described with particular emphasis on

plasmid R6K of Escherichia coli.

I N T R O D U C T I O N

Despite the great advances that have been made in our understanding of

mechanisms of DNA replication, there is little understanding at the molecular level

in any organism of the control of initiation of chromosomal DNA synthesis and its

linkage to the cell cycle. Because of experimental difficulties inherent in the genetic

and molecular analysis of the process of regulation of chromosomal DNA dupli

cation, considerable effort has been extended to the analysis of replication control of

bacterial plasmids. Plasmids, like the bacterial chromosome, are stably inherited -

i.e. the frequency of initiation of replication is regulated and at least for low copy

number plasmids a partitioning mechanism functions to assure that each daughter

cell receives one or more copies of the plasmid. The great utility of plasmids as model

systems for the control of initiation of DNA synthesis stems from their relatively

16 M. Fïlutowicz and others

small size, autonomous replication and dispensability for cell viability. It is evident

from studies to date that like bacterial and animal viruses, plasmids have evolved

different molecular strategies to assure their maintenance at a characteristic copy

number during cell growth and division. However, while different mechanisms are

employed to regulate the initiation of replication, in all instances where it has been

examined, plasmid replication control has been found to involve one or more

negatively controlling elements (Pritchard, Barth & Collins, 1969; Nordstrom,

1985). Furthermore, a majority of plasmid elements in Gram-negative bacteria

analysed to date utilize a system of replication initiation control that involves a series

of nucleotide sequence repeats (itérons) and a replication initiation protein (Filuto-

wicz et al. 19856 ; Chattoraj et al. 1985). This paper will concentrate largely on the

replication properties of this group of plasmids with particular emphasis on the

naturally occurring antibiotic resistance plasmid R6K that has been under study in

our own laboratory.

C O M M O N S T R U C T U R A L FE ATU RES OF P L A S M I D S C O N T A I N I N G IT ERONS

W I T H I N T H E IR R E P L I C A T I O N O R I G I N

Extensive analysis of the regulation of replication of plasmids ColEl, R I (FII) and

pT181 has demonstrated the central role of small RNA molecules that are specified

by these plasmids in the regulation of their copy number (for recent reviews see

Cesarini & Banner, 1985; Nordstrom, 1985; and Novick et al. 1985). The target of

these regulatory molecules in each case is a complementary RNA transcript. An

RNA duplex is formed and the resulting changes in the structure of the transcript in

each case leads to an inhibition of initiation of replication. For ColEl the target

transcript is the replication primer while in the case of R1 and pT181 the regulatory

RNA molecule interacts with the mRNA specified by the plasmid replication gene

resulting in a reduction of translation of the transcript and, therefore, a decrease in

the amount of the rate-limiting replication protein. While studies with these three

systems have greatly advanced our understanding of mechanisms controlling the

initiation of DNA replication and have been of fundamental importance in

establishing a regulatory role for small RNA molecules, these plasmid systems are

not representative of the majority of plasmid elements in Gram-negative bacteria that

are distinguished by possessing common structural features in the replication region.

This relatively large group includes R6K (Stalker et al. 1979; Stalker et al. 1982),

R485 (Stalker & Helinski, 1985), RK2 (Stalker et al. 1981 ; Smith & Thomas, 1984),

pSClOl (Churchward et al. 1983; Armstrong et al. 1984), F (Murotsu et al. 1981;

Tolun & Helinski, 1982), PI (Abeles et cil. 1984), Rtsl (Kamio et al. 1984), Adv

(Scherer, 1978; Grosschedl & Hobom, 1979) and RI 162 (similar or identical to

RSF1010) (Lin & Meyer, 1984; Haring et al. 1985). These plasmids contain direct

repeats varying from 17 to 24 bp in length within and/or near the replication origin

and in each case the plasmid specifies an essential and specific replication protein

(Rep protein). As illustrated in Fig. 1, these itérons are found as one or more clusters

and are either tandemly joined or separated by one or a few nucleotides. The start of

Repeat Size

19 bp

Regulation of plasmid replication 17

K M K h- H RE P l-<— 0-3 Kb-

P1 -*-()■! Kb-*>| R E P | ■* 0-2Kb- 19bp

PSCU)1 --- 0-2 K b — ^ 1 RE P I 18bp

R K 2 ^ --------------- (1-6 Kb -------------- = » ---------------------- 17bP

R 6 K .......................................... 0,3 K b ----------- » 1 REP I-------- 22 bP

Fig. 1. Repeats within plasnud replication regions. The position and orientation of

repeats are indicated by half arrows. Rep indicates the position of the structural gene for a replication protein. References for these structural features of the various plasmids are given in the text.

the Rep protein gene is generally a short distance (less than 200 bp) from one end of a

cluster of iterons. For Adv the origin is located within the O initiation protein gene

(Scherer, 1978) and in the case of RK2 it is likely that a transposon insertion is

responsible for the approximately 4 Kb separation between the iteron cluster and the

replication gene (Thomas, 1981).

R6K is a 38 Kb self-transmissible plasmid of E. coli that is maintained at

approximately 15 copies per chromosome. Electron microscopy analysis of R6K

replicative intermediates produced in vivo and in vitro has revealed three replication

origins designated, a, ¡3 and y, that are contained within a 4Kb region (Fig. 2)

(Lovett et al. 1975; Crosa et al. 1975; Inuzukae/ al. 1980; Crosa, 1980). In vivo the

a and [3 origins are used most frequently (45 % of the time for each) while in vitro

each of the three origins is used with equal frequency. Two components of the R6K

system are required for the activity of each of these origins; a 277 bp segment in the y

origin region that contains seven tandemly associated 22bp direct repeats (a HindlW

to Bglll fragment in Fig. 3) and a 35 Kd jt protein whose structural gene (pir) is

located between the a and ¡3 origins (Fig. 3). An eighth repeat and an inverted pair of

partial repeats is present in the pir promoter region (Fig. 3). Binding to the repeats in

the promoter region is involved in the autoregulation of ;r synthesis (Filutowicz et al.

1985<a; Kelley & Bastia, 1985).

Neither the a nor the ¡3 origin of replication is functional when separated from the

y origin sequences. DNA fragments containing the or origin will replicate only if they

are contiguous with the entire y origin region and if the j t protein is present (R.

Durland, unpublished observations). Similarly, the functionality of the ¡3 origin

requires the sequence from the Hindi 11 site within the y origin (Fig. 3) to well

beyond the end of the pir gene (Shafferman & Helinski, 1983). The conclusion that

18 M. Filutowicz and others

the itérons within the y-origin also are required for a functional a or ¡3 origin is based

on reconstruction experiments involving a y origin that is unable to bind n protein as

a result of mutations in the itérons. Neither a y plus a nor a y plus ¡3 replicon

containing these mutations is functional (M. McEachern, unpublished obser

vations). It has also been concluded that a functional ¡3 origin requires structural

integrity over much or all of the 1964 bp ¡3 origin region (Mukhopadhyay et al. 1986).

One of the reasons for this requirement is that a 17 Kd protein encoded by the region

adjacent to thepir gene (Shafferman & Helinski, 1983), designated the bis protein

(Fig. 3), is required for a functional ¡3 origin (Mukhopadhyay et al. 1986). This is

analogous to the results of recent studies on the requirements for replication initiated

from the two origins of mini-F plasmids (Tanimoto & lino, 1984; Tanimoto & lino,

1985). Three F plasmid specified proteins were found to be essential for the

replication from origin I but not from origin II in the mini-F plasmid. The presence

of more than one replicon with different requirements for replication in a plasmid

element may not be infrequent in view of the reports of secondary replicons in

Fig. 2. Physical and genetic map of plasmid R6K (Filutowicz et al. 19856). Hash marks on the circular map refer to the position of Hin d i l l sites. 4*,9 and 15 refer to H ind lll fragments present in the origin region, a, y and /3 indicate the locations of the 3 origins of replication, ter refers to the asymmetric terminus of replication. The positions of the streptomycin and ampicillin resistance genes are indicated by Smr and Apr, respectively. The single Bam HI restriction site is located by an arrow. The relative locations of the 7 direct repeats in the A origin and the 8th repeat in the operator-promoter region are indicated by dash marks.

Regula tion of plasmid replica tion 19

- on ex - (0-45)

ori yH vjO-lO) B

-------------- ori —>

(0-45)

B H H B

2000 bp

E-ORF-

y 0 280 1. 930 1290

S

2000 bp

✓ D X

Inc A////,-.Inc B |,: i : i ) *j|

pro

Jt Pro

600 bp

Fig. 3. Structure of the replication origin region of R6K (Filutowicz et al. 19856). The relative frequency of initiation from the a, y and ¡5 origins in vivo is indicated in parenthesis. A functional promoter region lies within the 7 direct repeats (designated the Inc A region) (P. Mukhopadhyay, unpublished observations). A 2nd incompatibility region (Inc B) lies between the 7 direct repeats and the 8th repeat. The putative promoter-operator region of the jz structural gene contains an inverted repeat indicated by facing arrows. The Jt structural gene and a 2nd open reading frame (designated bis) are present in the /3-origin fragment (Mukhopadhyay et al. 1986). HinA\\ \ andB^/II sites are indicated by H and B, respectively.

naturally occurring plasmids (Lane & Gardner, 1979; Sternberg & Austin, 1983;

Robinson et al. 1985).

P L A S M I D R E P L I C A T I O N P RO TEIN S B I N D TO ITER ON S IN THE R E P L I C A T I O N

R E G I O N

A positive role for the direct nucleotide sequence repeats in R6K replication was

first demonstrated with deletion mutants of the y origin that precisely removed one or

more of the repeats (Kolter & Helinski, 1982). Deletion of one or two repeats

resulted in a partially defective y origin and deletion of four or more of the seven

repeats inactivated the origin. A known requirement of the repeats is the binding of

the J t protein; demonstrated in vitro both with a fusion protein (Germino & Bastia,

1983a) and with the purified native dimeric form of J t (McEachern et al. 1985;

Filutowicz et al. 1986). It was found in this study that G to A transitions at both the

7th and 9th positions of either the first or the sixth repeat resulted in both a loss of

origin activity and an inability of J t to bind to the mutated repeat (McEachern et al.

1985). These two positions within each repeat are among those that are known to

interact with the jr-/3-galactosidase fusion protein and the native protein (Germino &

Bastia, 19836; M. Filutowicz, unpublished observations). It is of interest that the

positions of these mutations are contained within a consensus hexanucleotide

sequence that is present in each of the direct repeats within the replication region of

the F, RK2, Adv and PI plasmids (Filutowicz et al. 19856).

In addition to R6K, specific binding of a plasmid-specified replication protein to

iterons present within a replication origin has been demonstrated for Adv (Tsurimoto


& Matsubara, 1981; Zahn & Blattner, 1985), pSClOl (Vocke & Bastia, 1983), F

(Tokino et al. 1986) and PI (Chattoraj et al. 1984). Evidence that the binding of a

replication protein to the iterons at a replication origin results in an altered

configuration of this binding site comes from studies with X (Zahn & Blattner, 1985)

and R6K (Mukherjee et al. 1985). In both of these cases the electrophoretic

properties of the protein-DNA complexes suggested that the replication protein

induces bending of the DNA region associated with the protein. This protein-

induced bending may be necessary for the formation of specialized nucleoprotein

structures conceivably important for the control of initiation of replication (Echols,

1984).

THE I N I T I A T I O N P ROTE IN H A S BOTH POSIT I VE A N D N E G A T IV E ACTIVIT IES

AT THE R E P L I C A T I O N O R I G I N

When the J t protein, specified by R6K, is provided in trans a 400 bp sequence

containing the y origin is capable of replicating at the relaxed copy number

characteristic of the entire R6K plasmid (Kolter et al. 1978). Mutant J t proteins have

been produced that result in a loss of activity at high temperature (Inuzuka &

Helinski, 1978) or a change in R6K replicon copy number (Stalker et al. 1983;

Inuzuka & Wada, 1985; Filutowicz et al. 1986). As shown in Fig. 4 one of these

mutants, designated pir405-Cos (cold-sensitive), produces a substantially elevated

plasmid copy number in E. coli. The mutant plasmid in this case is a derivative of

R6K (plasmid pRK419) that contains the pir gene and the y and ^-origins of

replication (see Fig. 3). As shown in Fig. 4 the mutant phenotype is fully

complemented by a wild-type pir gene provided in trans. Other mutations near the

region of the pir405-Cos mutation in the pir gene have been shown to result in a j t

protein that produces a high copy number of a y or y plus replicon that is either

recessive or dominant in the presence of wild-type protein (Fig. 5). The properties of

thepir405-Cos and other trans recessive mutations in the J t structural gene indicated

that the J t protein plays a role in the negative control of R6K replication in addition to

its positive role for the initiation of replication.

A negative activity for the J t protein is also supported by three other lines of

investigation. Plasmid constructs containing either the wild-type or pir405-Cos

mutant gene downstream of a temperature inducible APR promoter were used to test

the effect of high intracellular levels of J t on the replication of a y-origin plasmid as

measured by pulse-labelling E. coli with 14C-thymidine (Filutowicz et al. 19856).

The results indicated that excessive levels of J t inhibited specifically the y-origin

plasmid pRK526 (replication of plasmids pACYC177, R300B, pBR322 and an RK2

derivative was unaffected). Similarly high levels of the mutant Cos405 jt protein were

much less effective than wild-type in inhibiting plasmid replication. Similar plasmid

constructs containing the pir genes downstream of a constitutive pBR322 promoter

were used to determine the effect of excessive intracellular levels of jt (8-fold over

normal R6K levels) in a transformation efficiency test in E. coli using plasmids

containing a y-origin alone, y and /3 origins, or all three R6K origins (Filutowicz et

15

III ori-7 Bgl III m i 111 I I n

Bgl II h i III on-( Bgl 11

Copy number of pRK419co.s405

42° 30°

82 221

NH, COOH

1 I__________________________________________________________ 1 <p 51 18 21

I---------------------------------------------------- — -------- 1 pAS752 17 22

C 2110 051

<N

%

Fig. 4. Copy number of pRK419 pir405-Cos in the presence and absence of wild-type Jr protein supplied in tram (Stalker et al. 1983;

Filutowicz et al. 1986). Relevant restriction sites are indicated within the pir structural gene. I l l and f refer to H in d ill and Hinil restriction sites, respectively. The/Zzradlll fragments of R6K present in pRK419 and pir405-Cos plasmids are designated 9, 15 and 2*.

The location of the Jt promoter is designated P^, whereas the eight 22 bp repeats are indicated by boxes, (p51 has n coding sequences

integrated into the C2110 host chromosome. pAS752 contains jr coding sequences inserted into the pBR322 derivative pAS260. TrpE

refers to the 7 N H 2-terminal amino acid residues of the trpE gene and the tryptophan operon promoter. The copy number of pRK419

pir405-Cos was measured in 10 ml cultures grown at 42°C and in cultures that have been shifted from 42°C to 30°C for 4h. Linearized

pRK248 was the internal standard for strain </>51. The lower half contains actual results of copy number determination of pRK419 and

pRK419 cos405 by gel electrophoresis and ethidium bromide staining of the plasmid band (lower).

Regulation

of plasm

id re

plicatio

n


—405 gly —» asp: 81 r r-13 leu—» arg:91 r (—120 ala —> val: 100 d r-116 pro—> leu: 106 d I" 200 phe—» ser: 107 d

PlrP H int

ATG — ■

HinP Bgl Hind Hind— TGA

Pir

Fig. 5. Position of amino acid substitutions as a result of copy-up mutations in the pir gene. The pir405-Cos mutation has been described previously (Stalker et al. 1983). The

identification and characterization of the pirl3 (M. McEachern), pir 116 and 120 (A.

Greener) andpzVZOO (P. Mukhopadhyay) mutants are unpublished.

al. 1987). The transformation results clearly indicated that an 8-fold overproduction

of the wild-type J t but not Cos405 protein specifically prevented the establishment of

an R6K derivative containing all three, two or a single replication origin. As controls,

plasmids pACYC177, pSClOl and RK2 were unaffected in their transforming

activity by these levels of J t protein. Therefore, by both tests, pulse labelling and

transformation efficiency, excessive levels of Cos405 protein were much less effective

than wild-type J t protein in the inhibition of replication of the R6K derivatives.

A third set of experiments demonstrating the negative activity of the n protein

involved plasmid RK2 constructs containing the pir gene with altered pir promoters

(produced by Bal31 nuclease digestion) that produced substantially less or more J t

protein than wild-type (R6K) in E. coli cells growing exponentially. As indicated in

Fig. 6 these constructs express J t protein over a several hundred fold range

(Filutowicz et al. 1987). Critical to this study was the development of a quantitative

immunoassay using a purified fraction of IgGs specific for the J t protein. Using

several different R6K y-origin plasmids to determine the effect of varying intracellu

lar J t concentration on copy number (as measured by Southern analysis with y-origin

specific radiolabelled probes) it is clear that J t protein at or near its (R6K) level

specifically reduce the copy number of y-origin plasmids and that J t levels

considerably higher than normal result in a substantial reduction of all three R6K

replicon plasmids tested. A second conclusion is that a J t level as low as 1 % of the

amount normally found for R6K is sufficient to maintain a y-origin plasmid at a

relatively high copy number. The data also clearly demonstrated that there is no

strict proportionality between the intracellular level of wild-type J t and the y-origin

plasmid copy number. In addition, the analysis of J t levels expressed by highly

transcribed pir mutants of R6K derivatives indicated that altered J t levels are not the

cause of the increased copy number in these cases - further demonstrating that the

total J t level is not rate limiting for initiation of replication from the y-origin of R6K.

Another unexpected outcome of this quantitative study is the estimation of 7000 to

20 000 monomers of the J t protein per cell. This is in contrast to the generally held

notion that an initiation protein is present in low and rate-limiting amounts.

It remains to be determined whether or not the replication proteins specified by

other plasmid elements exhibit both negative and positive activities at the origin of

replication. Overproduction of the PI RepA protein to levels well above normal has


PstlPIR

%

H A10 0-5-1

H A5 1-2

A14 2-5

A6 400-1200

, pPRl 100-200

RK2 Replicon

Fig. 6. Structure of wild-type and deletion derivatives of plasmid pPRl (Filutowicz et al. 1987). Thepir structural gene including its natural or altered promoter is inserted into an RK2 derivative (pTJS77). The extent of the deletion in each case is indicated by dotted lines. The level of Jt protein specified by each deletion is indicated as a % amount of wild-type (R6K) plasmid inis. coli.

been found to inhibit PI miniplasmid replication (Chattoraj et al. 1985). In addition,

PI plasmid mutants have been isolated that show a 5—8 fold elevation in copy number

and are recessive to the related P7 prophage (Scott et al. 1982). Finally, mutants with

similar properties to the R6K pir405-Cos copy-up mutant have been described for

the replication protein gene of a mini-F plasmid (Helsbergei al. 1985).

P L A S M I D R E P L I C A T I O N PROT EINS A U T O R E G U L A T E THEIR O W N S YN T H E S IS

Both in vivo and in vitro data have demonstrated that the expression of the R6K

pir gene is autoregulated (Shafferman et al. 1982; Kelley & Bastia, 1985; Filutowicz

et al. 1985a). Utilizing DNase I footprinting it has also been shown that the Jt

protein and E. coli RNA polymerase bind to the operator-promoter region of th epir

gene and that the binding sites overlap for three helical turns (Filutowicz et al. 1985).

The availability of the derivatives of plasmid pPRl described in the previous section

(Fig. 6) as well as a quantitative immunoassay for the K protein made it possible to

determine the minimal concentration of jz that is required for pir gene repression in

an exponentially growing culture (Filutowicz et al. 1987). As described in Fig. 7,

using a two plasmid system that consisted of the pPRl derivatives that produce

varying levels of n protein and a compatible plasmid (pRK775) that contains a pir

promoter fragment upstream of the /3-galactosidase gene as a transcriptional fusion, it


Ppir

+

RK2 Replicón

n Source Level of n p-Gal Activity

(% of wt) (units) (%)

None 0 1032 100

A10 <1 1970 190

A5 2 1770 171

A14 5 1440 140

A6-5 50 890 86

pPRl 150 122 12

Fig. 7. Effect of different K levels on the activity of the pir promoter (Filutowicz et al. 1987). Plasmid pPRl and its deletion derivatives were constructed by M. McEachern and are described in the legend to Fig. 6 and the text. Plasmid pRK775 consists of a transcriptional fusion of the pir promoter and part of the structural gene to the /3- galactosidase gene inserted into pBR322. Activity of /3-galactosidase and the level of jt was measured in exponentially growing cultures of E. coli M C I000. Relative n protein levels were determined by immunoblotting. The wild-type n level refers to the amount specified by plasmid R6K.

was found that to achieve a substantial reduction of p i r promoter activity the J t

concentration must be near the normal (R6K) level. In addition, it was observed that

levels of J t protein substantially lower than normal actually stimulated p i r promoter

activity as measured with this plasmid test system. These results suggest that the

major role of jt autoregulation is to prevent the production of excess concentrations of

J t that could result in a substantial reduction in copy number. It will be of interest to

determine the concentration dependence of repression of promoters of other plasmid

replication genes known to be autoregulated to determine if there is any similarity to

t h e p i r gene of R6K. Autoregulation of expression has been found for the replication

genes of PI (Chattoraj et al. 1985), F (Rokeach et al. 1985; Tokino et al. 1986) and

pSClOl (Linder et al. 1985; Vocke & Bastia, 1985 ; Yamaguchi & Masamume, 1985).

IT ERO N S A L S O P L A Y A D U A L R O L E IN THE R E G U L A T I O N OF R E P L I C A T I O N

The negative role of the direct nucleotide sequence repeats was first suggested by

plasmid incompatibility studies. The 277 bp Hindi 1 \-IigllI fragment of R6K

containing the seven repeats (Fig. 3) was found to be the major factor in the


H ® B pRK526 II M i n i n - I - , - f

0-3 Kb

^pBR322^ Compatible

H ©pRK419 H

i ipir

2 Kb

© H ©

+

pRK35 h

H vV B

I rTTrrTT1 I r

4 Kbpir

Incompatible pBR322 I (Exclusion of

R6K Replicons)

;

Fig. 8. Expression of R6K incompatibility by the direct repeats of R6K. The structure of the replication regions of plasmids pRK526, pRK419 and pRK35 is indicated by the relative locations of the direct repeats (boxes), the a, y and /3 replication origins, and the pir gene (S. Yang, unpublished observations).

expression of R6K incompatibility when various fragments of R6K were tested for

their ability to exclude R6K replicons when inserted into high copy number vectors

as shown in Fig. 8 (Filutowicz et al. 19856; McEachern et al. 1986). Mutants of the

R6K y origin region that are likely to contain multiple mutations in the iterons

recently have been identified and found to be greatly weakened in their incompati

bility (Inc±) properties (M. McEachern, unpublished observations). As judged by

the creation of extra 11 in d 111 sites most of these mutants appear to contain a G to A

transition at the 9th position of at least one of the iterons at the origin - a base

position previously demonstrated to be important for jt binding and origin activity

(McEachern et al. 1985). These results indicate that the R6K incompatibility

expressed by the iterons is related to their ability to bind jt protein - a finding that

supports the hypothesis that titration of jt protein by the direct repeats accounts for

the inhibition of R6K replication caused by excess copies of the repeats. A titration

model is also supported by in vitro studies demonstrating that high concentrations of

DNA fragments containing the iterons can inhibit R6K replication and the addition

of more jt protein reverses this inhibition (Inuzuka & Flelinski, 1978; Inuzuka et al.

1980). The titration of a plasmid-specified initiator protein has been proposed to

explain the incompatibility caused by direct repeats of other plasmids such as F

(Tolun & Helinski, 1981; Tsutsui et al. 1983), Rts 1 (Kamio & Terawaki, 1983) and

PI (Chattoraj et a l. 1984).

Regardless of the mechanism it is clear that iterons at or near the origin region of a

variety of plasmid elements exhibit a dual role in replication; they are required for

initiation to occur and they are capable of inhibiting it when cloned into a normally

compatible vector. 'Phis inhibitory role of direct repeats is likely to account also for

the observed increase in copy number when a non-essential cluster of iterons is


pMM31

V p M M 3 0

© (7)

Functional R6K yOR I

and pBR322

^,pMM33

pMM32

Fig. 9. Inserts of additional itérons in plasmids pMM3 and R6K. Plasmid pMM3

consists of a functional y origin represented by the seven boxes (direct repeats) between

the EcoRI (R) restriction sites and containing HindW I (H) and BglI I (B) sites. A

fragment containing six repeats was inserted at the £coR I sites in different orientations to

generate plasmids pMM30, 31, 32 and 33. The location of the single Bam (B) site in

plasmid R6K used for the insertion of R6K direct repeats is shown relative to the

positions of the a, y and ft origins (M. McEachern, unpublished observations).

removed from an origin region of a plasmid (Thomas et al. 1981) or mutated (Kline

& Seelke, 1982); or a decrease in copy number when extra copies of itérons are

inserted into a functional replicon (M. McEachern, unpublished observations). In

the case of the latter it has been observed that not only will additional itérons reduce

the copy number of an R6K y-replicon (illustrated in Fig. 9) but the degree of

reduction is dependent on the number of copies of the iteron inserted and the

position of the inserts (M. McEachern, unpublished observations; Lin & Meyer,

1984). A positional effect for the insertions is perhaps not surprising if there is some

level of cooperativity of binding of the jz protein to clusters of itérons when properly

spaced. Spatial restrictions on cooperative binding have in fact been observed for the

binding of A repressor molecules to adjacent pairs of operator sites (Hochschild &

Ptashne, 1986).

The sometimes contradictory facts concerning the dual regulatory roles of the Jt

protein and itérons of R6K may be best explained by a model for R6K replication

that is similar in certain aspects to that proposed by Trawick & Kline (1985) for the

regulation of F replication. According to this model, presented in detail elsewhere

(McEachern et al. 1986), the positive and negative functions of Jt are performed by

two biochemically distinct forms; an initiator form present at low levels and showing

strongest affinity to the itérons at the origin and an inhibitor form that makes up the

bulk of the approximately 10000 molecules per cell. This model requires a largely

irreversible conversion of each form to the other so that titration of the initiator form

by the direct repeats results in a decrease in the level of free initiator molecules that is

not compensated by relaxed autoregulation. The inhibitor form may inhibit R6K

replication by interacting or competing with the initiator form when bound to one or

more of the seven repeats.

Regulation of plasmid replication

ori /3---- ►

27

---ori y --- ►

----------- -------------------------- ----------------- 0

H B F B / \ H F Seal B

Hpal H

/ \12bp A (+) 58bp A (-)

234bp A (-)

267bp A (-)

Fig. 10. Binding of the it protein to sites within the replication region of R6K. The

relative positions of binding of the Jt protein to R6K are indicated by arrows. Both strong

(dark areas) and weak (dotted areas) regions of binding are indicated as well as the relative

positions of various restriction sites (H, HinAW \\ B, B glll ; F, F n u d ll ; H pal; and Seal).

The wavy line indicates the location of the pir gene. Inserts of D N A fragments at the

H pal and H in à lll sites either inactivate ( —) or have no effect ( + ) on /5-origin activity in

the presence of Jt provided in trans (Mukhopadhyay et al. 1986; Filutowicz et al. 1985;

Bastia et al. 1985; M. Filutowicz, unpublished observations).

P L A S M I D S M A Y FO RM A S P E C I A L I Z E D N U C L E O P R O T E IN STR UC TU RE

E S S E N T I A L FOR C O N T R O L OF IN I T I A T IO N OF R E P L I C A T I O N

Although the control of replication from the a and ¡3 origins of R6K appears to be

similar to that of the y origin, the mechanism by which it binding to the direct repeats

leads to replication initiation at the distantly located or and ¡3 origins (Fig. 3) remains

to be determined. As shown in Fig. 10, the finding that insertions and deletions of

extraneous DNA fragments at various locations within the 1964 bp ¡3 origin replicon

will inactivate this origin even if these intact regions are provided i n t r a n s

(Shafferman & Helinski, 1983; Mukhopadhyay e t a l . 1986) indicates a requirement

for structural integrity over an extended region for a functional ¡3 replicon. In view of

this structural requirement and the finding of J t binding sites distributed over a large

portion of the R6K replication region (Fig. 9) (Filutowicz e t a l . 19856; Bastia e t a l .

1985; M. Filutowicz, unpublished observations) it is conceivable that the controlled

initiation of replication at the ¡3 or a regions (and perhaps also the y origin) requires

the formation of a highly ordered nucleoprotein structure involving DNA sequences

in the entire replication region (a , y and ¡3) of R6K and the J t protein. There is

increasing evidence for a requirement of specific multiple-DNA protein interactions

for site-localization and regulated DNA transactions (Echols, 1984, 1986). These

interactions involving replication initiation proteins may serve to both bend (Zahn &

Blattner, 1985; Mukherjee e t a l . 1985) or fold (Dodson e t a l . 1985) DNA and

provide a nucléation site for the sequential loading of host replication proteins

(Dodson e t a l . 1985). Information on the formation and structure of specific

nucleoprotein structures involving plasmid replication regions may be a prerequisite


to a detailed understanding of the mechanism of control of replication of R6K and

other plasmid elements.

C O N C L U D I N G R E M A R K S

While plasmid elements display different strategies for the regulation of their

replication, many plasmids of E. coli, including R6K, share the property of

nucleotide sequence repeats at and/or near the origin of replication and a plasmid-

specified replication protein. The interactions between these iterons and the

replication initiation protein in the case of R6K involves both positive and negative

activities on the part of each of these essential components for R6K replication.

Considerably more information on the biochemical properties of the direct repeats

and the J t protein including their possible role in the formation of a highly ordered

nucleoprotein structure must be obtained before we can understand the precise

mechanism of regulation of R6K replication. As more information is accumulated on

the replication properties of the various plasmid systems, it should become clearer as

to whether or not the copy number of those plasmids with similar structural features

within their replication region is determined by a mechanism of regulation of

initiation that also is fundamentally similar.

This work was supported by grants from the National Institutes of Health and the National

Science Foundation.

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