effect of nusa protein on expression of the nusa, infb operon in e. coli

volume 13 Number 9 1985 Nucleic Acids Research

Effect of NusA protein on expression of the nusA,infB operon in E.eoH

J.A.Plumbridge*, J.Dondon, Y.Nakamura1 and M.Grunberg-Manago

Insthut de Biologic Physico-Chimique, 13 nie Pierre et Marie Curie, 75005 Paris, France, and'Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan

Received 2 January 1985; Revised 8 April 1985; Accepted 9 April 1985

ABSTRACTProtein and operon fusions between jacZ and various genes of the nysA.lnfB operonhave been constructed on A bacterlophages and used to show that the operon Isnegatively regulated by the level of NusA protein. Overproducing NusA (but not IF2)from a multicopy plasmld reduces the level of p-galactosldase from the fusionsIndicating repression of the operon. Introducing the A carrying the fusions into nusAmutant strains produces a higher level of 0-galactosldase-lndlcatlve of derepresslon ofthe operon. In particular, a larger form of the NusA protein which does not affectbacterial growth per se causes a derepresslon of the operon. As both protein andoperon fusions respond equivalently. we conclude that the nusA protein Is acting atthe transcriptional level to regulate expression of the nusA. infB operon.

INTRODUCTION

The NusA protein seems to be plelotroplc In the domain of transcription

termination. Its precise role jn vivo has still to be determined. The gene was first

identified by a mutation nusAI which prevented the growth of N dependent A

bacterlophage ( 1 ) . As AN protein Is required to overcome early transcription

termination during the lytlc cycle of A development, this phenotype Implied that the wild

type NusA protein was acting as an antltermlnatlon factor. The NusA protein was

subsequently Identified as the "L* factor necessary for the synthesis of p-galactosldase

In vitro (2) again Implying an antltermlnatlon function. As well as binding specifically

to AN protein (3) It Is also bound stolchlometrlcally by RNA polymerase core enzyme

( 4 ) . NusA protein bound to RNA polymerase can be replaced by slgma. The mutually

exclusive binding of sigma and NusA Implies that NusA acts at some point after

initiation.

Numerous jri vitro experiments support the Idea that NusA Is a transcription

termination factor. NusA was shown to enhance termination or pausing at the A tR2 site

( 5 ) . at the trpt terminator (0) and within the rrnB operon ( 7 ) . Evidence for a

termination function jn vivo was somewhat weaker ( 8 ) . Recently, however, amber

mutations In NusA have been Isolated on the basis that they allowed AN" phages to

grow ( 9 ) .

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Nucleic Acids Research

A reasonable interpretation of all these results Is that NusA Is primarily a

termination factor for RNA polymerase which is used by antltermlnatlon factors ( e . g .

xN) as a coupling protein to fix them to the transcribing polymerase complex. The

mechanism used by NusA to promote termination Is still obscure but It has been shown

to reduce the transcription rate In a sequence specific manner (10) and there is now

strong evidence that a particular DNA sequence called 'box A' is necessary for NusA

function during bacterlophage x transcription (11) and possibly for E. coll rRNA

operons (12 ) .

The nusA gene has been localised adjacent to InfB. the gene for translational

Initiation factor IF2 within a complex operon located at 69 min on the E. coll map (13 ) .

The first gene of the operon Is metY coding for a minor form of tRNAm* This gene Is

immediately preceded by a promoter shown Jn vitro (14) and |ri vivo (15) to be the

main promoter for the operon. The metY gene Is followed by DNA sequences

characteristic of two tandem rho-independent terminators (here called t-jtg) which

function in vitro (14 ) . After these terminators there is a series of structural genes, not

all of which have been identified with known proteins. A protein of sequence molecular

weight 15000 but with a mobility on SDS polyacrylamide gels of 21000 will be referred to

here as 15/21K and a second protein of molecular weight => 15000 will be referred to as

15k. The order of genes Is met Y, t i t P . 15 /21 . nusA. InfB. _15. (14. 15. 16. 17).

Further downstream, but transcribed in the same direction are the genes for ribosomal

protein S15 (rpsO) and polynucleotlde phosphorylaae (pnp) (18) . These genes seem

to have their own transcription signals (18 ) .

As nusA affects the transcription of various other bacterlophage and E. coli

operons It was pertinent to ask if NusA affected the operon of which It Is Itself a

member. As an approach to answer this question we have used the method of gene

fusions to make hybrid operons between nusA. InfB and lacZ using the vectors created

by Casadaban and coworkers (19) . We have used these fusions to Investigate the

effect of excess NusA on the operon and also to study the effect of a modified form of

the protein ; a hybrid between NusA and chloramphenicol transacetylase

(NusA: : cm). NusA does Indeed effect the level of expression of the whole operon and

In a manner characteristic of transcrlptlonal autoregulatlon.

MATERIALS AND METHODS

General

Standard procedures for recomblnant DNA work were taken from Manlatls et al

(20) or Davis et al ( 21 ) . Restriction enzymes and other DNA modifying enzymes were

all from commercial suppliers and used as recommended. General bacteriological

3372

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Table 1 E.

Kane

a) E.coli

IBPCS321

IBPC3341

IBPC535S

IBPC5357

IBPC181

rBPC183

JC3560

IBPC242

IBFC241

IBPC244

IBPC246

IBPC252

IBPC251

IBPC2S4

IBFC2S6

coll and Bacterlophage x strains used

Relevant Genotype

F~ thi-1, argG6, argE3, hls-4, «tl-l,

xyl-5, tax-297, rpaL, AlacX74

IBPC3321 recA

IBPC5321 nusAiten

IBFC53S5 recA

IBPCS321 arot; , nusA( tal)

IBPC183 his4, recA

F~ argG6, natBl, hia-1, leu-6, mtl-2,xyl-7, malAl, gal-6, lacTJ, tonA2,

tsx-l,supE44, pnpiiTn5, X

JC3560 ar^G , nusAiicm, pnp

JC356O nusAiten, pnp

JC3560 argG .nusAticn

JC3560 nusAiicn

IBPC242 mal+, X°

IBPC241 mal+, x"

IBPC244 «al+, X*

IHPC246 mal+, x"

b) Bacterlophage x

AHM540

XSEH

X512

X512Lac~

Xnav8-5

XE2

XF7

XD7

UBB21, cl+, nlnR

CI857, nlnS, W4O3 , E11OO ,3100

A(exo, bet, gam, lnt), CI857, lac

A<exo, bet, gam, lnt), CI6S7

1DB21, cl , nlnR, pheS,T-lac fusion

lm21, cl , nlnR, ln£B-lacZ protein fusion

Isn21, cl , nlnR, nusA-lacZ protein fusion

lnB21,cI,nlnR,15/21-lacZXX operon fusion

Origin orReference

pheS , argG

derivativeOf IBPCSB01 (23)

This work

•

m

M

Pt

(24)

nils work

la

M

M

-

PI

m

m

(25)

(26)

(27)

nils work

(28)

This work (Figl)

nils work

mis work (Flg2)

techniques are mostly described In Miller 122) as Is the method for assaying 0 -

galactosldase.

The E, coll and bacterlophage x strains used In this work are listed In Table I.

pBP280 (24) Is shown in Figure I. it carries a 10 kb insert Including argQ, the njjsA.

3373



A 18 21kA ar«Q matY I nu»A 11

\

rp«O pnp

TflH E

psnsoft lac'Z Y A Sa

PMC1403

lm*rt E-Bfl ol p8P2S0 Into pWC1403 batwaan E and B $ DMAF2 (Lac*)E Bo/B Sa . E

pMAFl

nutAHacZ hybrid (Lac«)

tnaart E-Sm ol pBP280 Mo pMC1403 batwa«n E and Sma$pMAE4 (Lac*)E Sm B 8a E

I „,„„ K I I P*IA1!4

Waaat with Bam HL fffl In with Hanow, r»fioata^pMAE4-B (Lac*)E Sm 8a EI lnr./»y/»uiiJ ~1 I

mm-tacZ hybrid (Lac*)M HSS.7 M

J I I |att | Imm21

Sa

lacZE Sac

PMAE4-S

Xna*S-S

\512Lac"

DMAE4-Bz - W B nu»A Otga«t \na«8-5 wtth M«tD, AS12 Lac'wtth EcoR1hybrid J | and PMAE4-B wHh EcoR1 and Mat D, Koala, In

H E B vitro packagaI t:i!!iy/V//HI 1 attl N CI857

E(S3.8%)Sac/ WA»7d j ltt] H CI8S7

lacZ' 'lacz;tE

±Am

Sa

'lacZHnfB nu«A|m«tY c)gOhybrid IS/21k

In vivo Croat with ANMMOdmmZD

- f alt N knmti \E2

1kb

F IQ . 1 Derivation of xE2 carrying an InfB-lacZ protein fusion.Tho looatlon of genes Identified on pBP280 Is Indicated. Only the relevant restrictionsites are shown. Details of the construction are given In Materials and Method. %refers to the position of the equivalent sites as percentage of wild type A. A primepreceding or following a gene means that the gene Is Incomplete on that side.E = EcoRI. H = Hlndlll. B = BamHI. Bg = BgJII. 8m «• Smal. Sa = SaJI. M » Mstll.Sao = Saol

InfB operon and rpaO and pnp. Plasmlds of the nusA. InIB region are listed In Table 2.

and shown In Figure 3 together with the genes oarried. pB19-l Is the IO. 2 Kb left hand

part of pBP280 from EcoRI to jHlndlll. pB2O-l carries the 5. O kb Sail to BgJII

fragment. The nusA gene is not complete on this plasmld but the slightly truncated

protein Is stable and functional. (pB2O-1 is equivalent to the pYN87 ( 9 ) ) . pB22-4

3374



Table 2 Plasmlds of the nusA.lnfB region and the relative Intracellularconcentration of NusA and IF2 In strains carrying them.

Plaomld

pACYC184

PG9-3B

pG9-3ARuBA

PBR322

JDB19-1

PB20-1

PB22-4

PB23-18

PB23-11

pB18-l

Genes of the(1)

nusA,infB region

P, B>etY,t 1.15/21 ,nuaA,in£B

P, metY,t,t_,15/21,lnfB

argG, p, metY, t , t,,, 15/21, nusA, lnfB

argG,P,metY,tt ,15/21,nuflA1

P,netY,t,t,,,15/21,nuflA'

P,metY,t,t,,15/21

P.metY.t t ,

nusA,infB

Relative

NUBA

1

5-8

0.9-1.5

1

6-11

(3)3-6

n.d.

n.d.

n.d.

1-1 .5

( 2 )Levels of Protein

IP2a

1

7-10

7-12

1

9-20

0.6-1

n.d.

n.d.

n.d.

1.5-3

IP20

1

6-10

6-10

1

6-16

0.6-1

n.d.

n.d.

n.d.

2 - 3

(1) The ONA Insert carried by these plasmids Is shown in Figure 3. P refers to themajor promoter for the operon and t^g to the tandem rho-lndependant terminators.(2) The cellular levels of NusA and IF2 were measured in exponentially growingcultures of IBPC5321 XF7. IBPC5321 xE2 and IBPC5341 AD7 carrying the variousplasmlds. Extracts were analysed by electrophoresis on duplicate SDS poly-acrylamide gels which were blotted onto nitrocellulose and treated with antl-NusA orantl-IF2 followed by « i | protein A as described (34) . The treated blots wereautoradlographed and the radioactive bands were cut from the blot and counted toquantltate the amount of NusA or IF2. These values were normalised to the proteinconcentration In the extracts. The amount of crude extract analysed on the gelsvaried from 1 to 10 /ig. Relative levels of NusA and IF2 were calculated by comparingwith the strains carrying only the vector plasmld. The numbers show the range ofvalues obtained over the three cultures.(3) Mixture of wt protein and slightly smaller truncated proteinn.d. = not determined

carries tho 1. 9 kb Pstl fragment from the beginning of the nusA. lnfB operon. (and Is

equivalent to pKUl (14) and pSMll (15) ) . This plasmid produces a smaller NusA

fragment than pB2O-l. which still complements the nusAI mutation (14) but not the

3375Downloaded from https://academic.oup.com/nar/article-abstract/13/9/3371/2381742by gueston 04 April 2018


conditional amber mutants (15 ) . pB23-l l and pB23-18 are derived from pB22-4 by

deleting between the 1st or 2nd Pvul sites of the operon and the Pvul site of pBR322.

pB23-11 carries metY and part of 15/21 whereas pB23-18 carries metY. all of 15/21

and 14O bp of nusA. The construction of pQ9-3B and pG9-3ANus will be described

elsewhere. The E. coll Insert of both of these plasmlds starts at the Pstl site

Immediately In front of the principle promotor of the operon and ends at the Hjndlll site.

pG9-3ANus carries a deletion of 1.3 kb Internal to nusA which leaves the genes for

15/21 and InfB Intact. The derivation of pB18-l . which Is missing the principle

promoter of the operon. has been previously described (29 ) .

The construction of protein fusions between nusA or InfB and lacZ carried by x F7 and x

E2

a) Construction of protein fusions on plasmids : A nusA-lacZ protein fusion was made

by Inserting the EcoRI to BgJII fragment of pBP280 Into pMC1403 cut with EcoRI and

BamHI as shown In Figure 1. pMC1403 derived plasmlds (amplcillln resistant (Am r ) )

which had received the pBP28O fragment were identified by the presence of the argG

gene on the cloned fragment (IBPC5321 Is argE and arqG but arqQ+ strains can be

detected by growth on cltrulllne). Comparison of the nusA DNA sequence (16) with

that at the beginning of lacZ carried by pMC14O3 (19) predicts that the two proteins

will be In phase at the hybrid Bglll/BamHI site. In agreement with this the resultant

plasmlds e .g . pMAF2 (Fig. 1) gave blue colonies on Xgal (Xgal is 5-bromo 4-chloro

3-indolyt p galactoslde) containing plates.

An InfB-lacZ protein fusion was made in an anlogous way by Inserting the EcoRI

to Smal (using Xmal as enzyme) fragment of pBP280 into pMC1403 cut with EcoRI and

Xmal. The InfB sequence at the Smal site is not in phase with lacZ (17) but the phase

could be corrected by a +1 (or +4) frameshlft. The Lac~ plasmid (pMAE4) was cut at

the BamHI site, which was filled In with DNA polymerase (Klenow fragment) and

rellgated to give Lac+ plasmlda of the type of pMAE4-B (Fig 1) .

Maxloell analysis of the proteins encoded by the pMAE4-B fusion plaamld

showed two high molecular weight protein bands (of about 14O and 16O kDaltons)

corresponding to the two forms of IF2. a and 0. fused with p-galactosldase and wild

type NusA protein (30 ) . The pMAF2 plasmid produced a single high molecular weight

band of about 160 kOaltons corresponding to the hybrid nusA-p-galactosldase

protein (data not shown).

b) Transfer of the protein fusions to x : A convenient way to transfer lac fusions from

plasmlds to bacterlophage x Is to make use of the xs. derivatives of xplac5. which

carry lacZ next to the structural genes of the left arm (e. g. see 2 1 . 27) . Several "rare"

restriction sites exist In lacZ which can be used to cut both plasmid and x vector In

order to recreate the lacZ gene on the recomblnant x. As the EcoRI site at the end of

3376



lacZ Is mutated In pMC14O3 (19) we used instead the Mstll site near the beginning of

lacZ. This process is diagrammmed In Figure IB for pMAE4-B. The pMAF2 fusion was

transferred to x In exactly the same way.

The EcoRl to Mstll fragment carrying the fusion was Inserted between the left

arm of Anav8-5. a Lac*. I m m " phage (28) up to the Mstll site In lacZ and the right

arm of A512 (27) up to the EooRl site at 53. 8 %. A512 Is a Lac* cl857 phage carrying

a deletion between BamHI sites at 57. 7 and 71. 0 % A. To facilitate distinction between

the A512 starting phage and the required Lac+ fusion. A512 was made Lac" by

Inverting the Sac I fragment which covers the lacZ-A Junction to give A512 Lac~. pMAF2

or pMAE4-B were digested with EcoRl and Mstll. Anav8-5 with Mstll. and A512 Lac~

with EcoRl. The llgated mixtures were packaged In vitro and Lac+ Imm* cl857 phages

selected by plating on IBPC532KANM540) on X gal containing plates at 37°C. LacZ*

(blue) phages were purified on IBPC5321. Phages which carried arqO ware detected

since they formed lysogens of IBPC5321 at 3O°C which grew on minimal plates

supplemented with citrulllne and histldlne. Small scale liquid lysates (23) of argQ+.

Lac'1' phages were grown and phage ONA extracted. Phages of the type xpMAE4 (Fig

IB) and ApMAF2 were characterized.

These thermosensltlve lmmA cl857 ]nt phages were ohanged to I m m " . c l * . int+

phages by In vivo recombination with ANM540(25). A permissive host. IBPC5321. was

Infected with both phages at an mol of 2-3 . allowed to grow for 2hr and then plated on

IBPC5321 (A+> on Xgal containing plates to select for Lao+ I m m " phages. Such

phages were purified on IBPC5321 and LacZ*. arqG+ lysogens selected. These were In

turn purified and the phages Induced by U. V. Phage ONA was prepared and AE2 (InfB-

lacZ fusion) (Fig IB) and AF7 (nusA-lacZ fusion) Identified, by restriction enzyme

analysis.

Construction of a promotor proximal operon fusion carried by AD7

a) Construction of an operon fusion vector with a Pstl site : The BamHI to Sajl Jac

operon cassette of pMC871 (19) was cloned between the BamHI and Sail sites of

pGA39 (31) (chloramphenlcol resistant (Cm r ) ) to give pQMl as shown In Figure 2.

This produced Cmr colonies which were blue on Xgal. despite the absence of a

functional Lac promotor (pMC871 in also blue). The extraneous DNA between the Saml

and BamHI sites of pGMl was eliminated by digesting with Xmal and BamHI. filling In

the sites with DNA polymerase. Klenow fragment, and rellgatlng to give pGMA. This

process recreates both the Smal and BamHI sites which now overlap by one base pair.

The presence of this plasmid causes colonies to be blue on Xgal containing plates and

medium red on MacConkey lactose plates (350 units of p-galactostdase)

b) Insertion of a promotor proximal fragment of the nusA-lnfB operon : The 570 bp

Pstl-Xholl fragment of pB23-11 (Fig 3) was Inserted between the Pstl and BamH 1 sites

3377



I trp'BA lacZ Y A I* Km i p M C 8 7 1

j ..-•'' ln»art BamHI-Sall ot pWCB71 Into

pGA39

P Sm BCut pGM1 with B«mHIand Xmal,

S» E P fin In wtthKlanow, Bgata^pGalA

l™* r t P l t I " X h ° n o l P 8 * ' 1 1 woPB23-11 pOtlA<8fl««te<! wtthP«tIandBamHI

htrp'BA lacZ Y A

Sa E

EIA43.8-S3.8) So<67.5, 68.5%)I «tl | | N CI857

Ugaat \SEW with Sail,

\NMS40 with Psti, and

PQM01 with Psti and Sail.

UO«t«, In vitro packaga p sB

S^oct Lac'lmm21 I

* \D7

«t t

\SEW

P<66.S%)I N Imm21 \ N M 5 4 0

| , c A Y Z trpA B1 j P

PQM01

E(A43.8-53.8)

I attSa

11C A Y Z I

hybf Id oparon) Xn^y

lmn.21 XD7

'15/21k

1kb

FIQ. 2 Derivation of XO7 carrying a promoter proximal operon fusion(see legend to Fig. 1) . In addition P ° Psti. Xh = Xholl

of DGMA. This fragment carries the major promotor of the operon. metY. the two

terminator structures and half of the gene for 15/21K. pQMA derivatives which had

acquired a promotor containing fragment could be identified as those which were now

very strongly red on MacConkoy lactose plates. Such plasmids were analysed and

pQMOl was Identified (Fig 2 ) .

c) Transfer of the operon fusion to x : The Pstl-Sall fragment of pGMDl was Inserted

into the clll region of X between the Psti site at 06. S % and the Sail site at 67. 5 %. In

XNM540 the I m m " substitution removes the Psti site at 76. 3 % x . . leaving the Psti site

at 60. 5 % as the last site, x SEW (20) was digested with Sail. xNMMO with Psti and

pQMDl with Psti and Sajl. The DNAa were Iigated and packaged In vitro and then plated

on IBPC532KX*) on Xgal containing plates at 37°C. Blue plaques were purified on

3378



15/21

E

II

matv/ nusA tnfBA. I /?9 I

I f^j^^

AntibioticReaistanc*

Am

AmCmCm

TcTcTcAm

parts of the

Replicon

PBR322

PBR322pACVCpACVCPBR322D6R322PBR322PBR322

nusA-.Fig. 3 Structure of a series of plaamlds carrying parts of the nusA-. infB operon.The location of identified genes of pB19-l Is Indicated. The fragments of ONAsubcloned in the other plasmids Is shown. These subclones are further described InMaterials and Methods. The genes carried and the level of NusA and IF2 producedaro described In Table 2. Replicon pACYC means that these plasmlds carry thesame replicon as pACYC184 whloh is present in the vector plasmid pGA39(31) usedfor trto cloning.

IBPC5321 and lyaogens. picked from the center of the plaques, were purified and

Induced by U. V. Phage DNA was prepared and AQMD7 Identified. Experiments with this

phage were subsequently conducted in recA strains because the construction includes

a small duplication of about 1 % A ONA on either side of the insert which could be lost

by In vivo recombination.

Construction and characterization of a nusA-cat fused gene

Close and Rodriguez (32) have described the construction of chloramphenlcol

resistant DNA cartridges carrying the structural gene for chloramphenlcol trans-

acetylase (cat) but missing the natural promoter. The cat cartridge with Bam HI sites

at the ends was inserted into the unique BpJII site at the end of nusA of pBP280 (Fig 1)

so that the cat gene is transcribed In the same sense as nusA, This plasmid now

confers Cmr to the host bacteria. In addition to the vector encoded Tc r .

This plasmid (pBP280 Bojll: : cm) was introduced Into strain JC3560. which

is argQ and carries a Tn5 (Kanamycln resistant. Kmr ) transposon within pnp

(24 ) . The transformants were purified once and then grown through several cycles of

serial dilutions In L8 and then In LB containing chloramphenlcol. The saturated

cultures ware plated on the Tc8 selection plates of Maloy and Nunn (33) . After 2 days

growth at 37°C. Isolated colonies were picked and screened for ArgQ. Cm r . Kmr and

Tc r. Analysis of the Tc8 bacteria. I .e. those which had lost the plasmid. showed a

large number which had acquired Cmr with or without the adjacent Arg and Km

markers. One example of each pattern of recombination was further analysed : -

IBPC 241. 242. 244. 246 (see Table I ) .

3379



1 2 3 4 5 6

w.t.NusA-*~

Fig. 4 Immunoblott analysis of Cmr recomblnants.Freshly saturated cultures were lysed by boiling In 3DS sample buffer and analysedon a 10 % 8D8 polyacrylamlde gel (35 ) . Proteins were electrophoretlcally blottedonto nitrocellulose, treated with antlNusA and then protein A labelled with « i | (34)Lane I. JC3S60 : Lane 2. IBPC244 : Lane 3. IBPC246 : Lane 4. IBPC241 : Lane 5.IBPC244 : Lane 6. control bacteria (maxlcell strain CSR 603).

JC 3500 Is xr due to a malA mutation but It and the Cmr derivatives were

converted to \a by phage PI tranoductlon selecting for growth on maltose. The

recomblnants all grew normally and plated K* (an N dependent phage) normally at

42°C I. e. There was no phenotype like the nusAI mutation of Friedman and Baron

t l J ,

Samples of each recombinant were analysed by immunoblottlng (34) with antl

NusA as shown in Figure 4. All (our recomblnants showed a band reacting with antl

NusA antibodies of a molecular weight greater than wild type NusA. Maxlcell analysis of

the pBP280 BoJII: : cm plasmld similarly shows a band of molecular weight about BO

kDaltons. (data not shown). Comparison of the ONA sequence of nusA at the BoJII site

(18) with that of the beginning of the cat cartridge (32) shows that nusA Is fused in

phase with the cat gene such that the 30 nucleotldes before the natural Initiation codon

of cat are translated as 10 additional aminoaclds. The 80 000 daltons molecular weight

protein band is thus NusA (up to the BgJII site) plus 10 amino acids plus the 24 000

daltons of the chloramphenlcol transaoetylase protein. Maxlcell analysis of pBP280

Bp.Hl:: cm also showed considerable synthesis of wild type molecular weight chloram-

phenlcol transacetylase. thus we cannot say whether the hybrid NusA:: cm protein has

CAT activity or whether the Cm r derives from the wild type protein. The hybrid protein

would appear to have NusA activity since the recombinants are perfectly viable. The

Immunoblott (Fig 4) does show two faint bands somewhat smaller than wild type NusA.

so It is. in theory, possible that It Is these fragments rather than the hybrid NusA: : cm

which is permitting growth.

3380



The nuaA: : cm mutation was introduced Into IBPC5321 by phage PI trans-

ductfon of argG* or cm r from IBPC 252 (direct selection for Cm r was very Inefficient

compared to selection for argQ*) IBPC5355 was a rare transductant obtained by

selecting directly for Cm r which Is still argQ. It was converted to recA by conjugation

with an Hfr strain carrying recAl and selecting for His'*'.

The rujsA(tsl) mutation was introduced Into IBPC5321 using Pi grown on

YN2351 (Nakamura. Misusawa. Tsugawa. Zubar. Court. Imai. manuscript in prepa-

ration) and selecting for ArgQ+ at 30°C to give IBPC181. The recAl allele

was subsequently introduced by conjugation as for IBPC5355. to give IBPC183.

RESULTS

1. Description of nusA-lnfB fusions with lacZ

Two types of fusions between genes of the nusA-JofB operon and jac have

been constructed and transferred to Integration proficient >. bacterlophage. The first

type are protein fusions between nusA and lacZ (XF7) or InfB and lacZ (AE2) where

an N terminal fragment of NusA or IF2 is fused In phase with 0-galactosldase at the

level of the 8th amlno acid of the wild type 0-galactosidase protein to produce hybrid

NusA-0-galactosidase or IF2-0-galactosldase proteins. In the case of the InfB-lacZ

fusion carried by XE2 (Fig 1 ) . two hybrid proteins corresponding to the two forms of

IF2. a and p. are synthesized (30 ) . The 0-galactosldase activity of these fusions is

a measure of the level of transcription of the operon and of the translation of the

nusA or InfB mRNA. These fusions carry the major promoter of the nusA. InfB

operon plus several Kb. Including argQ. upstream of the operon.

The second type of fusion constructed Is an operon fusion carried by xD7

(Fig 2 ) . A short promoter proximal part of the nusA-lnfB operon. lacking any part of

the nuaA or infB structural genes, has been cloned before a lacZ complete with its

own translatlonal initiation signals but missing a functional promoter. In this case

wild type 0-galactosldase Is synthesized from the transcription signals at the

beginning of the nuaA-lnfB operon.

2. Overproduced NusA protein decreases expression of the nusA-infB operon

The effect of a series of plasmlds (Table 2 and Fig. 3) covering various

genes of the nusA-lnfB operon has been tested on the level of 0-gaiactosldase

expressed from the three fusions. The results are shown In Table 3. At the same

time the level of NusA and IF2 produced by these plasmids was measured by

quantitative Immunoblottlng and the results are shown in Table 2. Only plasmlds

which cause an appreciable overproduction of NusA protein (pQ9-3B. pB19- l .

pB20-l) have any effect on the level of p-galactosldase from any of the fusions.

They reduce the 0-galactosldase activity to about 50 % of the wild type activity. On

3381



Table 3 Effect of NusA and IF2 overproduction on the level of expressionfrom different nusA. InfB-lac fusion

Fusion type

Plaamid

PACTC194

PG9-3B

PG9-3ANUSA

(SR322

PB19-1

PB2O-1

JB22-4

PB23-18

PB23-11

PB18-1

XD7

15/21-lac operon

1 0 0

32HO

8314

1 0 0

3014

5712

7 0

108

1 0 5

9416

D

( 2 )

( 2 )

( 2 )

( 2 )

( 2 )

( 2 )

( 1 )

( 1 )

( 1 )

( 2 )

• 4 "

42

6 0

4 5

3 6

6 5

6 0

32

35

35

35

XP7

NuaA-Lac protein

. * «

100

55*17

9615

100

4314

5316

8812

10314

94

93*6

)

( 3 )

( 4 )

( 3 )

( 4 )

( 3 )

( 3 )

( 3 )

( 2 )

( 1 )

( 2 )

• 4 "

36

42

40

37

SO

48

33

34

33

33

IP2-Lac

' * "

1OO

72110

9614

1 0 0

6218

7612

8812

9812

9 1

97118

XE2

protein

)

( 4 )

( 4 )

( 3 )

( 4 )

( 4 )

( 2 )

( 2 )

( 2 )

( 1 )

( 2 )

35

40

33

35

48

33

40

33

40

33

Monolysogens of IBPC9321 with XE2 or XF7 and IBPC5341 with X07 were transformedwith the series of plasmids listed. Cultures were grown at 37°C In MOPS mediumenriched with all amino acids (36) and containing the relevant antibiotic : Cm (25/igml"1). Tc (10 Mgml"1). Am (500 MOml"1). Further Am was added during growthto maintain the presence of the plasmids.(1) 0-galactosldase activities were measured for each culture at. at least, fourpoints between A e 5 0 values of 0.2 and 0. 6 and are expressed as a percentage ofthe strain carrying the vector plasmld. The numbers in parenthesis are the numberof Independantly grown cultures tested. For IBPC534KXD7) 100 % = 1540 i 50units, for IBPC 5321 (XF7) 100 * = 360 i 40 unite and for IBPC5321 (XE2) 100 % =325 i 20 units.(2) D j Is the average doubling time In mln for each culture.

the other hand, plasmids which overproduce IF2 in the absence of NusA have no

effect on the fusions, compare pQ9-3B and pG9-3ANus. The plasmld pB18-1.

which carries the genes for both nusA and InfB but without the major promoter

causes a barely measureable Increase In NusA concentration (Table 2) and has no

effect on j3-galactosldase expression from the fusions. Qualitatively similar results

are observed for all three fusions tested. Quantitatively the IF2-Lac protein fusion

carried by xE2 seems to respond less than the promoter proximal operon fusion

3382



Fin. 5 Effect of growth rate on £-galactosldase expression from xE2 and AF7.IBPC 3321 lysogenlsed with AE2 or AF7 was grown In minimal MOPS mediumsupplemented with different carbon sources (36) to produce a range of growth rates: 1) Acetate : 2) Pyruvate : 3) Qlucose : 4) Qlucose enriched with all the amlnoaolds (36) n Is the number of doublings per hour. The results are the mean of sixmeasurements of 9-galactosldase activity between Agjo values of 0 .2 and 0.5 (orA 0.1 - 0. 25 for acetate).

(XD7) or the NusA-Lac protein fusion. (XF7) this point Is considered In the

Discussion.

As the promoter proximal fusion responds In the same way to the effect of NusA

as does the nusA-lac protein fusion It Is probable that the main effect of NusA is

transcriptlonal. Overproducing a large N terminal fragment of NusA from pB2O-1

causes almost as much repression as does wild type NusA. The somewhat smaller

fragment synthesized from pB22-4 seems also to exert some represser effect. The

plasmlds carrying the promoter proximal genes metY and 15/21 have no effect on the

level of 0-galactosidase. We. however, have no proof at the moment that these

plasmlds cause an augmentation In the cellular level of their gene products. Thus any

effect might be masked If their expression from the plaamids Is already severely

repressed.

As shown in Table 3. the presence of the plasmlds which overproduce NusA and

IF2 do reduce the doubling time of the rysogenlc bacteria. It was thus possible that the

decrease In 9-galactosldase seen was due to a decrease In growth rate rather than a

specific repressor effect. The level of 0-galactosldase under different growth rates was

measured and Is shown in Figure 5. Increasing the doubling time does decrease the

level of 0-galactosldase. However increasing the doubling time from 30 to 60 mln (the

range observed In Table 3) produces only a 15 % decrease In p-galactosldase activity

and not the 50 % decrease observed. In the experiments described above.

We also tested the effect of the plasmlds which overproduce NusA on wild type

9-galactosldase synthesized from the chromosome and saw none. Chromosomal lacZ*

strains carrying pQ9-3 or pG9-3 ANusA. induced with Isopropyl thlogalactoslde (IPTQ)

3383Downloaded from https://academic.oup.com/nar/article-abstract/13/9/3371/2381742by gueston 04 April 2018


Table 4 The effect of mutant alleles on the level of expression from differentnusA. InfB-lac fusions

Fusion type

Strain

IBPCS341

IBPCS357

IBPC183

Genotype

w.t.

nusAitcm

nusA( t s l )

XD7

15/21-lac op

100 ( 2 )

169*2 ( 3 )

183*5 ( 3 )

eron

°T 2 >

82

80

88

X

nusA-lac

1 0 0

160*1

176*16

Yl

Z protein

)

( 3 )

( 3 )

( 3 )

7 0

7 0

8 0

XE2

infB-lacZ

1 0 0

120*2

133*3

protein

D

( 3 )

( 3 )

( 3 )

r2>

72

7 0

7 0

Isogenlc derivatives of IBPC5341 carrying the nusA: : cm or nusA(tsl) allele werelysogenlsed at low m.o. l . with the fusion phages. Lysogens were grown In MOPSmedium supplemented with arglnlne and hlstldlne at 30°C.(1) 0-galactosldase measurements were made for each culture at four pointsbetween A^SQ = 0.2 to 0.5 and are expressed as a percentage of the wild typestrain. The number In brackets Is the number of Independent monolysogens tested.For IBPC5341 (XF) 100 % Is 325 t 55 units : for xE2 100 % = 345 ± 40 units and forXD7 (TOO <*>1 1700 * 65 units.(2) Or Is the average doubling time In minutes for each culture.

gave £-galactosldase values of 106 % and 112 % compared to strain carrying

pACYC184. We can thus eliminate the possibility that overproduced NusA non-

speclflcally reduces gene expression. We conclude therefore that the repression is

specific although the magnitude is not very large.

3. Mutated NusA proteins derepress the nusA-lnfB operon

Two mutant forms of NusA protein were testsd. The modification of NusA protein

by addition of the 24 kDaltons of protein corresponding to the gene for chloramphenlcol

transacetylase Is described In Materials and Methods. The gene for this hybrid protein

was Introduced by homologous recombination, onto the E. coll chromosome. It

seemed to exert no appreciable effect on the bacteria except that the cells are now

chloramphenlcol resistant. The second mutated nusA gene Is the nusA(tsl) mutation.

This thermosensltlve mutant. Is defective In transcription termination at both 30°C and

42°C (Nakamura et al. . manuscript In preparation).

The two mutants IBPC5357 (nusA: : cm) and IBPC183 (nusA(tsl)) and the

Isogenlc parental (IBPC5341) were lysogenlsed at low m.o. l . with the three fusion

phages XE2. XF7 and XD7. ^galactosldase measurements were made on several

Independent lysogens to Identify monotysogens. Table 4 shows that both the nusA: : cm

and nusA(tsl) mutations cause a dereprasslon of all three fusions. The magnitude of

the effect Is very similar for the xO7 and xF7 fusions (160-180 %) but Is somewhat less

for the XE2 fusion (120-130 % ) . This Is not surprising though, since xE2 carries a wild

3384



type nusA gene which can complement the nusA mutation. Measurements In Table 4

were made at 30°C because of the thermosensltlvlty of the nysA(tsl) mutation. The

strain was also tested at 37°C. where It still grows normally, but no greater

derepression was observed.

DISCUSSION

The results reported here show that the level of functional NusA protein in the

cell affects the level of expression of the operon. Such a result is characteristic of an

autoregulated protein. The phenomenon has been Investigated using both operon

fusions and protein fusions with lacZ. Numerically similar results were obtained with

both types of probes and as the operon fusion used carried only the proximal part of the

nusA. InfB operon and none of the structural gene for nusA it seems very likely that the

autoregulation Is occurlng at the transcrlptlonal level.

The structure of the nusA. InfB operon suggests at least one possible

mechanism for how this autoregulation might occur. The first gene of the operon metY.

coding for a structural RNA. tRNA ftf . is followed by sequences characteristic of two

rho-independant terminators (14 ) . It is only after these terminators that the genes for

the structural proteins of the operon ( 1 5 / 2 1 . nusA. InfB. 15) are found. One role of

these terminators is presumably to reduce the level of transcription of the proteinMetgenes which follow, compared to the amount of tRNAj required. Thus NusA

could be regulating the amount of readthrough of these terminators. Several other

terminators have been shown to be sensitive to NusA protein. (5. 6. 7) . Alternatively

NusA has been shown to slow down transcription or promote pausing at sites not

recognisable from their sequence as terminators (7 . 10) and so it is possible that the

effect of NusA on Its own operon acts outside the terminators, but within the first 570 bp

of the operon cloned In AO7.

In any case the effect of NusA. whether direct or Indirect. Is felt throughout the

operon as far as the InfB gene. The InfB protein fusion does, however, seem to be less

sensitive to the overproduction of NusA protein than the earlier fusions. One possible

explanation for this Is that the minor promoter detected just In front of InfB (15. 29) Is

functioning In this fusion to enhance InfB expression when upstream transcription Is

reduced.

The magnitude of the repression effect Is not very great. A 7-10 fold

enhancement In NusA concentration produces only a 50 % decrease In gene

expression. It was thus possible that the results observed were due to other effects

e. g. : an effect of NusA on Initiation of transcription cannot be eliminated at this point.

One hypothesis tested was that the presence of the plasmlds causes a reduction In

growth rate and hence a reduction In the level of 0-galactosldase synthesized. The

3385



data of Figure 5 shows that reducing growth rate does decrease expression from the

fusion but not as much as the decrease observed with the plasmlds which cause NusA

overproduction. Another hypothesis, for which there Is no evidence, would be that

NusA controls the expression of something else which acts In trans to regulate the

transcription of the nusA. InfB operon.

Looked at from the other point of view, the fact that any change in the

expression of 9-galactosidase from the fusions Is observed on changing the growth

rates suggests that the nusA.lnfB operon is subject to "so-called" metabolic

regulation. The change observed is not very great though. An eight-fold Increase In

growth rate produced only a 60 % increase In p-galactosldase activity from x£2 or XF7.

Comparable experiments using genes fused to 0-galactosldase or galac-

toklnaae have shown for rrnE about lOO % increase for a less than three fold

Increase In growth rate (37) : for tufB a 600 % Increase In 0-galactosldase for a

fourfold increase In growth rate (38) and for trpS a 250 % Increase for a four fold

Increase In growth rate (39 ) . For these latter experiments, it was concluded that the

gene under study was subject to metabolic regulation. The much smaller effect seen

for the nusA-lnfB operon might imply that although basically subject to metabolic

regulation, the effect Is reduced by other regulatory factors coming Into play.

The nusA: : cm mutation described here Is interesting In the sense that the

protein does not appear to have any detectable phenotype on cell growth. However It

does exert a regulatory effect in enhancing Its own expression, and that of IF2

(Nakamura. Plumbrldge. Oondon. Qrunberg-Manago - manuscript In preparation)

and that of the nuaA-lacZ fusions described here. The magnitude of the derepresslon

seen is comparable to that observed with the nusA(tsi) mutation whioh has both a

strong thermosensltlve phenotype and Is defective In termination (Nakamura et al. .

manuscript In preparation). The nusA: : cm mutation has not been tested for

termination efficiency. It is possible that the protein is sufficiently defective In

termination to enhance expression of the operon but not to affect cell growth.

One Interpretation of the results described here is that the C terminal part of

NusA could be the region of the protein Important for regulation. The fact that from the

Bglll site onward 37 amino acids can be replaoed by protein sequences of

chloramphenlcol transacetylase shows that these sequences are not necessary for

NusA function, at least as far as permitting normal cell growth. However, this region

could be important for regulation as the effect of this mutation on the fusions is

comparable to that observed with a defective nusA mutation (nusA(ts l ) ) .

An alternative and maybe preferable hypothesis Is that the regulatory part Is

associated with the N terminal region. The protein synthesized from pB20- l . which Is

mlislng the same 37 amino acid* that are replaced in nusA:: cm. represses nusA. InfB

3386



operon expression almost as effectively as wild type NusA. The distinctly smaller

protein synthesized from pB22-4 (missing 150 amlno acids) also seems to exert some

repressor effect. These observations are not necessarily contradictory with the result

that the nusA: : cm mutation derepresses the operon, since It Is quite conceivable that

adding 24 kOaltons to the C terminal end of the protein causes a staple modification In

the N terminal region.

IF2 synthesis Is co-regulated with that of NusA. The precise reason for this is

still obscure. IF2 synthesis Is co-ordinated with that of the other translational Initiation

factors IF1 and IF3 and also to ribosomes (40 ) . How this co-ordination is achieved Is

not known. For IF3 at least two transcription start sites have been Identified (41 ) . For

IF2 a second minor promoter In front of the InfB gene has been claimed (15, 29) . This

second promoter could be functioning In the case of repression of the nusA.lnfB

operon (see above). These multlmodal transcription units could thus be involved In the

co-ordinated synthesis observed, but much more work Is needed to unravel this

particular aspect of cellular regulation.

To summarize, the data reported here strongly suggest that the NusA protein

autogenously regulates Its own transcription and that of the neighbouring genes. There

is as yet no definite evidence for a mechanism but It is tempting though to postulate that

NusA regulates transcription through the tandem terminators near the beginning of the

operon.

ACKNOWLEDGEMENTSWe thank Martin Schmidt for the gift of antl-NusA antibody. We thank Scott Butler forcritical reading of the manuscript and Mathias Springer for constructive discussions.This work was supported by grants to Doctor Qrunberg-Manago from the "C. N. R. 8."(Grant 18) and from the I .N .8 . E.R. M. (823.008. 831.003 and a short termfellowship granted to Y. Nakamura). from the ' M . R . I ' (82 V 1289). from the'FONDATION POUR LA RECHERCHE MEDICALE' and "E. I. du Pont' (to M. Q . - M . ) .

"Present address: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box6666, 260 Whitney Ave., New Haven, CT 06511, USA

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3388


effect of nusa protein on expression of the nusa, infb operon in e. coli

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