Control of phenylalanyl-tRNA synthetase genetic expression

J. Mol. Hiol. (1985) 184, 3144

Control of Phenylalanyl-tRNA Synthetase Genetic Expression Site-directed Mutagenesis of the pheS, T Operon Regulatory Region in Vitro Jean-Franqois Mayaux’ , Guy Fayat’, Michel Panvert’, Mathias Springer2 Marianne Grunberg-Manago and Sylvain Blanquet’ 1Laboratoire de Biochimie, Ecole Polytechnique 91128 Palaiseau Cedex, France 21nstitut de Biologic Physico-chimique 13 rue P. et M. Curie, 75005 Paris, France (Received 10 December 1984, and in revised form 13 February 1985) Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant Ml3 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a 1 phage carrying a pheS, T-1acZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 basepairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in t’he translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

1. Introduction

All these genes are carried by an EcoRI-Hind111 fragment of the E. coli chromosome, which was cloned in pBR322 to give plasmid pB1 (Plumbridge et aE., 1980). The genes pheS and pheT are adjacent and transcribed from the same promoter (Plumbridge & Springer, 1980). The DNA sequence of the pheS, T operon and transcription experiments in vitro (Fayat et al., 1983) strongly suggested that this operon could be regulated by a phenylalaninedependent attenuation mechanism very similar to that described in the case of several amino acid biosynthetic operons (reviewed by Kolter & Yanofsky, 1982). This regulatory mechanism involves controlled transcription termination at a site, the attenuator, located in the leader region

The genes pheS and pheT code for the two subunits of phenylalanyl-tRNA synthetase, a tetrameric enzyme of the azpz type (Fayat et al., 1974). They are located at 38 minutes on the standa,rd Escherichia coli map (Comer & Bock, 1976) and belong to a cluster of genes all involved in the translation process. The order of these genes was shown to be thrS, infC, rplT, pheS and pheT, all transcribed in the same direction from thrS to pheT (Plumbridge et al., 1980; Plumbridge & Springer, 1980). The genes thrS, infC and rplT code for threonyl-tRNA synthetase, initiation factor 3 and ribosomal protein L20, respectively (Springer et al., 1977; Hennecke et al., 1977; Fayat et al., 1983). 0022-2836/85/130031-14

$03.00/O

31

0 1985 Academic Press Inc. (London) Inc.

32

J.-i?

Mayaux

between the transcription start site and the first struct,ural gene of the operon. The pheS, T leader transcript. (or attenuated transcript) carries a. potential peptide-coding region specifying a 14.residue peptide containing five phenylalanine residues. The model initially proposed by Lee & Yanofsky (1977) for the trp operon of E. coli, states that translation of such a peptide-coding region would regulate the formation of alternative mRNA secondary structures, one of which is recognized by RNA polymerase as a transcription termination signal. Transcription termination at the attenuator (attenuation) is thus controlled by the extent of aminoacylation of tRNAPh” with phenylalanine. In the case of t,he pheS, T operon, this mechanism is further supported by studies in vivo (Springer et al.. 1983). The pheS, T operon is derepressed whenever the intracellular concentration of aminoacylated tRXAPh’ is decreased or the translational properties of tRSAPh” are affected. The latter point was demonst,rated using a miaA mutant that, cannot ensure isopentylation of the adenosine 3’-proximal to the anticodon of several tRh’As, including tRNAPh’ (Eisenberg et al., 1979). This type of is known to relieve transcription mutation termination at several other attenuators including trp and pheA (Yanofsky & Siill, 1977; Gowrishankar & Yittard, 1982). More recently, a &-acting mutation that lowers pheS, T transcription about, sixfold and abolishes

et al.

the attenuation control was characterized as an IS4 element insert,ed in t,he t.erminat,or stem of the pheS. 7’ at.tenuator (Mayaux et al.. 1984). In addition, several deletion mutations in the regulatory region of the pheS, T operon were and their obtained effects on downstream confirmed expression the location of the transcription signals determined in vitro (Springer et al., 1985). In particular, these studies showed t#hat the transcription terminator of the attenuator was essent,ial to the regulation of the operon. In this study. further mutations (deletions. insertions and point mutations) were introduced in the control region of the pheS, 7’ operon. Sit,e-, direct,ed insertions or deletions were obtained using limited heteroduplex formation of recombinant 3113 vectors. Point mutations were isolated using sodium bisulphite as a mutagen (Shortle & Botstein. 1983). After characterization. the different mutations were introduced on 3. phages carrying pheS. T-la& operon fusions. These phages were used to lysogenize suitable lZ. coli strains and the effect of mutations on the regulation of pheB, 7 expression could be measured conveniently by t,he level of fl-galactosidase activity in t,he cells. The results of this study are discussed in relation to the location of t,he mut,ations wit,hin the m RNA st,ructures and the leader peptide secondarj sequence”c’omprising the pheS, T at8tenuator region.

Table 1 Strains

used in this zcork Reference and/or origin

E. coli strains

Relevant genotype

.JMlOl

A(lac-pro). supE, thi, (F’traD36. lUCZAM15)

11~PC115(R+)

F- A(lar-pro), rLal.4, rpoB, metB, urgEam. .supE, ara. rrcA 1, 1’. lysogenized with I+

$1. Springer: IHP(:I 15 is a multistep derivative of XA103 (Coulondre & Miller. 1977)

IBP(‘I 11

I-- A(/ac-pro), nald, rpoB, m&B, urgEam. supF, am, rrcA1. I.‘. 1-

RI. Springer: multistep derivatiw of XA102 (Coulondre & Miller. 1977)

proAB, lac P,

Messing ( 1979)

II. Springer: multistep derivatives of Y I and 1’2 respectively (Tanofsky 8r Still. 1977) Plasmid and phages

Storable markers AmpR, TetR. thrS, infcl, rplT, pheS, pheT, pUR322 derivative Ml3 derivative carrying a set of unique restriction sites EcoRI, SmaI, BarnHI, SalI, P&I, Hind111 in the beginning of kc& X-terminal fragment

Plumbridge

it af. (1980)

Messing 8r Vieira ( 198%)

Same as mp8 but opposite polarity of the above restriction sites; carries an a.dditional HaelI fragment derived from pSR322 imm%l. cl+, ninR

Messing & Vieira (lSS2j

~18.57, nin5, rplT, phd, T-lae fusion inzmdl, cl + ninR, pheS, T-lac fusion

Springer et al. (1983)

13orck et ctl. (19%) This work (Fig. 4)

Mutagenesis

of

2. Materials and Methods (a) Strains and

general

techniques

The E coli strains, Ml3 and A bacteriophages. and the plasmid used in this work are listed in Table 1. General genetic techniques were as described by Miller (1972) and Davis et al. (1980). The Ml3 vectors M13mp8 and M13mp9 (Messing & Vieira, 1982) were used with the host E. coli strain JMlOl following experimental procedures described by Messing (1983). Phage 1 DNA was prepared according to Yamamoto et al. (1970) and Maniatis et al. (1982). pB1 plasmid DNA was obtained as described by Fayat’ et al. (1983). Lambda recombinants were packaged in vitro according to Hohn (1979). For rapid screening of Ml3 and 1. recombinants, small-scale Ml3 replicative form (Birnboim & Doly, 1979) and I phage (Davis et al., 1980) DNA preperations were used. Lambda phages for small-scale DKA preparations were grown on supplemented LB agarose plates (Arber et al., 1983): plat’e lysates usually yielded titers of about 10” plaqueforming units/ml. For restriction analysis, 1 DSA was further extracted with phenol to remove any residual agarose. Lysogenizations of YlRl and Y2Rl strains with the IX phages were performed by infecting bacteria at a multiplicity of infection of 0.1. After preadsorption for 15 min at 37°C. about lo4 cells were plated on RMM X-gal plates (Springer et al., 1983). Several blue colonies were purified and tested for resistance to 1 imm21 cland sensitivit,y to 1 vir. For each lysogenization, t,he fi-galactosidase activity levels of several (4 to 7) independent clones were measured. These levels varied as multiples of the lowest value that was considered to reflect fi-galactosidase expression from a monolysogen. In our conditions, 769,; of analysed clones were monolysogens, 17% dilysogens, 4oj, trilysogens and 30, polylysogens. j3-Galactosidase activity was assayed with toluene-treated cells as described by Miller (1972). (b) Deriva,tion of M13X8BS

and M13X9BS

The recombinant phage M13X8BS was constructed in 2 steps. A 1052 bpt Sau3A-Sau3A fragment (Fig. 1) overlapping both rplT and pheS genes (Fayat et al., 1983) was separated by agarose gel electrophoresis from a complete Sau3A digests of the 14.58 kb pB1 plasmid. After electroelution of the corresponding band from the gel (Maniatis et al., 1982), the fragment was cloned int,o the unique BamHI site of M13mp8. When inserted in the right orientation. a recombinant phage with the struct.ure of M13X8BS could be obtained readily by deleting the DNA between the SmaI site located in front of pheS and the unique SmaI site of M13mp8. The resulting phage MlSXSBS carries a 439 bp Suu3A-SmaI fragment containing the end of rplT and the intact, pheS, T regulatory region, between the BumHI site (which is reconstituted) and the SmaI site of M13mp8. This fragment was then transferred into M13mp9 to give M13XSBS. After digestion of M13X8BS with BamHl and EcoRI. the fragment was separated by electrophoresis, electroeluted and cloned between the BamHI and EcoRI sites of M13mp9 (Fig. 1). Throughout this work? the precise structures of 3113 recombinants were verified systematically by restriction analysis of the replicative form DNA and by DNA

t Abbreviations pairs.

used: bp, base-pair: kb, lo3 base-

33

pheS, T Attenuator

sequencing of the single-stranded viral DK,4 using the dideoxynucleotide chain termination method of Sanger et al. (1980). (c) Derivation

of M13XZ3, Ml3XIIl

ad

Ml3XA2

As described in Fig. 2, M13X9BS single-stranded DI\A (18 pg) was hybridized to 50 ng of the 17-mer “universal” sequence primer (PI, Biochemicals) in 50 ~1 of 10 mMTris. HCI (pH 7.5). 10 mM-k&Ci2, 50 rn>q-XaC’1 during 15 min at 55°C. After slow cooling to room temperature, limited synt’hesis of t,he minus strand was carried out using a limited concentration of dCTP. Thr W-PI reaction volume contained 20 mill-Tris HCI (pH 7.5). 0.7 mM1.4-dithioerythreitol, 7 miv-MgCl, and 35 mw-KaCl. The DEA was incubated at, 30°C for 1 min wit.h O-2 mM each of dATP. dGTP and TTP. and 30 units of the Klenow fragment of E. coli DNA polymrrase I (Boehringer-

regulatory pheSJ region OhiSl PBI du3A

.&a1

Sab3A

Ix

or/ JGCI- /ocZ’

II

M13mp8

M13X 80s

Ml3mp9

Ml3X9BS

Figure 1. Isolation of M13X8BS and M13X9BS. Only the relevant parts of pB1 plasmid of Ml3 phages and the most important restrictions sites are shown. .4 complete restriction map of this region of pB1 DXA is given by Fayat et al. (1983). rpZT, pheS and pheT are. respectively. the genes for ribosomal protein L20, the small and large subunits of phenylalanyl-tRNA synthetase. pheSl is the presumed leader peptide whose translation regulates transcription of the pheS, T operon t,hrough the attenuation mechanism. The small arrow represents the - 150 bp attenuated transcript. the sequence of which is given (as the DEA image of the antisense strand) in Fig. 4. Genes II and IV are Ml3 genes. ori is the intergenic DNA region carrying the origins of plus and minus strand DNA synthesis. la& and Ia&’ are t.hr C-terminal and N-terminal fragments of Zac repressor and fi-galactosidase genes, respectively, carried by the 789 bp fragment from the E. coEi Zac operon found in M13mp8 or mpg. M13X8BS was isolated in 2 steps as described in detail in Materials and Methods, section (b).

J.-F. Mayaux HinfI BornHI

et al.

SRhI

H//If1

smo1

:

Ml3 x I II replicative form

DNA

sinple-stranded viral DNA

’ Transform

115

Ml3 x D9

A f

1

’ XhoI

x XhoI

I

\

* Transform

t

\

BglII

XhoI

delefmn

HSO; v RF-DNA/XhoI * Hybridize

with

* Eisultite * DNA-polymefose

MiEX9BS SS-DNA 1

* Tron:torm

Figure 2. Diagram operon. Experimental (m) XhoI linker; (IJ)

illustrating procedures unmodified

the strategy for generating point mutations in the regulatory region of the pheS, 7 are described in detail in Materials and Methods. sections (b) to (f). (a) SphI linker: insert; (@) remaining DNA after digestion with BaE31 exonuclease.

Mannheim; 9000 units/mg); after addition of 10 PM-dCTP. the reaction mixture was further incubated for 30 min at 30°C. The DP;A was recovered by precipitation with et.hanol and digested with 9 units of HinfI during 1 h at. 37°C in medium salt digestion buffer (Maniatis ct al., 1982). The reaction mixture was then extracted with phenol, extracted with ether, precipitated with ethanol and re-treated with the Klenow fragment of DEA polymerase I under the same conditions as described

above. except for the addition of 0.1 rn~-d(‘W and 0.2 mM-[cx-32P]dATP (0.6 Ci/mmol) to trace the DKA. After precipitation with ethanol, portions of DKA were either directly circularized or ligated in t.he presence of phosphorylated &&I linker (5’ dG-G-(:-A-T-C:-(‘-(’ 3’: Roehringer-Mannheim) using the standard pro(sedurrs described by Maniatis et al. (198”). Among plaques obtained after transfection of JMlOl. 10 were picked. purified and used to inoculate 1 ml caultures of ,JMlOl.

35

Mutagenesis of pheS, T Attenuator The phages were propagated during 5 h and singlestranded DNAs prepared. Phages were initially screened by DNA sequencing with the universal primer and running only the dideoxy-guanosine-terminated products on a sequencing gel with the M13X9BS control DNA. About 50’& of the clones carried the desired modification. i.e. (1) a filled-in HinfI site in the pheS, T control region and (2) the insertion of one sph1 linker in that site. One clone was further characterized in the case of each modification. Complete sequencing using the 4 reactions identified phages with the structures of M13XI3 and M13XIlI. respectively (see Fig. 5). M13X.42 was obtained using a similar procedure. After limited synthesis of the second strand, the DNA (5 pg) was digested by 3 units of Hue11 (Boehringer-Mannheim) during 1 h at 37°C and subsequently treated with 10 units of phage T4 DNA polymerase during 30 min at 37°C in a 70.~1 reaction volume containing 30 mw-Tris-acetate (pH 7.9), 60 mM-potassium acetate. 10 mM-magnesium acetate, 0.5 mM-1,4-dithioerythreitol, 0.1 mg bovine serum albumin/ml and all 4 deoxynucleotide triphosphates at a concentration of 0.1 mM each. After precipitation with ethanol, the deleted DNA was ligated and used to transfect, JMlOl. Clones were analysed using the screening procedure described above. (d) Isolntion

of Ba131 deletions around the unique Sphl

siteof M13XIlI

SphI-restricted MlRXIll replicative form DNA (20 p(g) was treated with 1.2 units of &z/31 exonuclease (Bethesda Research Laboratories) 30 s at 30°C in a 200.~1 reaction volume containing 12 miv-CaCl,, I2 mM-MgCl,, 0.2 M-NaCl, 20 mivf-Tris. HCl (pH 8.0) and 1 mr+EDTA. The reaction was stopped by adding EGTA (pH 8.0) to a final concentration of 15 mM. The DNA was extracted with phenol, extracted with ether, precipitated with et’hanol and repaired using the Klenow fragment of DNA polymerase I. The DNA was then ligated in the presence of XhoI phosphorylated linker (5’ dC-C-T-C-G-A-G-G 3’: Biolabs) and used to transfect JMlOl: 25 plaques were purified and inoculated into 1 ml cultures with JMlOl. Replicatire form DNAs of the phages were prepared using the small-scale procedure of Birnboim B Doly (1979). The clones were screened for the presence of the XhoI linker and t’he size of the deletion was roughly estimated by restriction analysis. Nine phages were selected and their single-stranded DNAs were prepared and sequenced. (e) Construction of unidirectional deletions: derivation MlSXDSL, M13XD9R, M13XD2R and Jfl3XD4R

oj

In order to isolate unidirectional deletions around the site using the bidirectional deletions obtained above. the phage M13XI15 was constructed: SphTrestricted M13XIll was treated with T4 DNA polymerase to remove 3’-overhanging ends and a XhoT linker was inserted in the blunt site (another phage, named Ml3XI7, was obtained by ligation of the blunt site without added linker). Replicative form DNAs of M13XI15 and of the phage carrying the selected bidirectional deletion were digested with XhoI and BglI (see Fig. 2). Both fragments from bot,h phages were separated by agarose gel electrophoresis and electroeluted. The small fragment of M13XI15 was t,hen ligated with the large fragment of the deleted phage (or vice aersa) and used to transfect JMlOl. By this method. phages were obtained, the DNA of which HinfI

contained only the left or the right part of the original RaZ31 deletion (M13XD9L, D9R. D2R. D4R). When desired, BarnHI-EcoRI inserts carrying monodirectional deletions were transferred into M13mp8 as described in sect’ion (b), above. (f) Isolation

of point

mutations by bisulphite mutagenesis of M13XP812, P846, P845, P843, P837, P817, P914, P924, P909 and P942

in vitro. Derivation

Derivatives of M13X8BS carrying one or several point mutations were isolated essentially as described by Everett & Chambon (1982). A portion (28 gg) of wildtype single-stranded DNA (M13X8BS or M13X9BS) was hybridized to 4pg of XhoI-cut replicative form DNA of the appropriate deletion mutant (M13XDSL. D9R or D4R) in a 20-~1 reaction volume containing 10 mMTris. HCl (pH 75), 1 mM-EDTA and 0.2 mM-NaCl. After 3 min in boiling water, the hybridization mixture was left for 6 h at 65°C. slowly cooled to room temperature and precipitated with ethanol. The DNA was redissolved in 0.1 x SSC (SSC is 0.15 M-NaCl, 0.015 M-sodium citrate. pH 7) and treated with 3 M-sodium bisulphite (pH 6.0) for 1 h at 37°C in the dark in the presence of 0.5 mMhydroquinone. Dialysis treatment of the DNA samples was as described by Shortie & Botstein (1983). After precipitation with ethanol, the DNA was treated with 10 units of endonuclease-free E. coli DNA polymerase I (Boehringer-Mannheim) during 3 h at 18°C in the nicktranslation buffer (Maniatis et al., 1982) containing the 4 dNTPs (0.2 mM each). ilbout 0.7 pg of DNA was then used to transfect JMlOl. In each experiment. 30 to 50 clones were purified and analysed by DNA sequencing as described in section (c), above. and in Fig. 3. In our conditions, only the cytosine residues exposed in the single-stranded gap of the heteroduplex reacted with bisulphite (3 M, 1 h). Out of a total of 145 analysed clones. 25”/b were unmodified phages (i.e. carried the wild-type sequence of M13XSBS or M13X9BS), 27qi, had a single point-mutation, 22% had 2 mutations. 15% had 3 mutations, 3% had 4 mutations and 194 had 5 mutations. The remaining clones (about 7%) either carried unwanted modifications or corresponded to the original deletion mutant. Ten single or multiple point mutations were selected for further characterization (MI3XP812 to M13XP942 in Fig. 5). (g) Derivation

of AX phages

The AX phages were constructed by inserting the SmaI-Hind111 fragments of the M13X phages (carrying the pheS, T regulatory regions with various deletions, insertion or point mutations) between the left arm of iMT,P (up to the first SmaI site in front of pheS at 53.8% of i+) and the right arm of 1NM540 (from its Hind111 site at 56.7% of i’, Fig. 4). The 1ML2 DNA was digested by SmaI. the iNM540 DNA by Hind111 and the M13X by both enzymes. The restricted DNA9 were mixed in equimolar amounts, ligated and packaged in vitro. The packaged lysates were titered on RMMX-gal plates with IBPC 115 (A’) as indicator strain. The indicator strain was chosen to be immune to I immA infection to eliminate any reconstit’uted phage carrying immi.. Several blue plaques from each cloning were purified twice on TBPC 115 (lb+) and twice on IBPC 111. Phages were grown on agarose plates (see section (a), above) with TBPC 111 as indicator strain and DNA from lysates was analysed by digestion with restriction enzymes. For each mutant. phages with the structure of i.X were identified

36

et al

J.-F. Mayaux

Ml3

X 86s

t

I T

C

G

A

I

2

3

4

5

6

7

Fig pre 3. A sequencing gel used in the screening of point mutants afher mutagenesis with bisull white. The dideo Nci,v c: termi nation s of 7 clones are Ishown in tracks 1 to 7 next to the complete sequencing of “wild-ty ipe” M13X8BS The esidu es, indicate ,d b\ deleti on muItant M13XD9L (I.erloned in M13mp8) was used in this experiment. Twelve cytosine 1-1 arroR iheads on the right side cif the gel, were exposed to sodium bisulphite in the single-stranded ga,p. Tl le numberi ng of bases is the same as in Fig. 5. The strand sequenced here is the strand complementary to the viral strar 1t1of M13X .8BS.

(Fig. 5). The 1X phages are imm21 integration proficient phages. They carry a pheS, T-lacZ fusion identical to that of 1ML2. recombined at the SmaI site with different mutated pheS, T regulatory regions,

3. Results (a) Isolation

and characterization of mutations pheS, T control region

in the

(i) Construction of insertion mutants A 439 bp SauSA-SmaI fragment of plasmid pB1 carrying

the end of the rplT

structural

gene and the

regulatory region of the pheS, T operon (see Fig. 1) for site-directed was used as a substrate mutagenesis of the pheS, T attenuator region in

vitro. The SauSA-SmaI fragment was cloned between the BamHI and SmaI sites of Messing’s vectors M13mp8 and M13mp9. As this pair of vectors contain the same set of unique restriction sites with opposite polarities in the lac Dlr;A (Messing & Vieira, 1982), the fragment was inserted in opposite orientations in the resulting phages named M13X8BS and M13X9BS. In order to create either deletions or insertions in the attenuator region, we used the unique Hid site of the insert. This site overlaps the terminat,ion codon of the presumed leader peptide sequence. Since there are 27 other Hid sites either in the lac or Ml3 sequences, a method was designed in which only the cleavage site located in the insert could be This method is based on the cut by Hi&I.

Mutagenesis of pheS, T Attenuator SmoI

H/rldlTT

37 I kh

Mutated M13X

XML2

XNM540

XX

Figure 4. Construction of 1X phages. Only the relevant part of I phages and essential restriction sites are shown. ?;, means percentage of 1+. A prime preceding or following a gene symbol means the gene is incomplete on that side. J, xiu, int are A genes. infC and thy&’ are, respectively, the genes for initiation factor 3 and threonyl-tRKA synthetase; the deletion between the 2 genes in IzML2 is shown in black. trp strands for D?U’A internal t,o trp operon. The detailed construction of the pheS, T-la& fusion of 1ML2 is shown by Springer et aE. (1983). The horizontal arrow under ,$X represents the hybrid transcription unit that is created by this fusion. The expression of fi-galactosidasr from the pheS, 7’ promoter in the YlRl and Y2Rl strains is shown in Table 2 and in Fig. 5.

properties of the HinfI restriction enzyme, which is specific for double-stranded DNA (Waye et al., 1983; and our unpublished results). The “universal” sequence primer was annealed to the single-stranded viral DNA of M13X9BS and limited synthesis of the minus DNA strand was performed. In this reaction (see Materials and Methods), one of the nucleotides (dCTP) was present, at a limiting concentration such that polymerization stopped after the incorporation of about 400 nucleotides in the nascent strand. Since the Hinf’I site of the insert is located 205 nucleot’ides 3’ to the primer and is separated by 457 nueleotides from the next Hinfl site in lacl’, only the Hinff site in the attenuator was recognized and cut (see Fig. 2). After circularization in the presence or absence of &‘$I linker, the mutant phages M13XI3 and M13XIll (see Fig. 5) were isolated and characterized. M13XI3 carries a filled-in Hinfl site, which corresponds to the insertion of the trinucleotide AAT, two base-pairs after the stop codon of the leader peptide. The M13XIll phage contains an 1I-nucleotide sequence (AAT plus the 8-nucleotide SphI linker) inserted at the same position. Using MlSXIll, two other insertion mutants were constructed. MlSXIll replicative form DNA was cleaved by SphI and the 3’ protruding ends (5’ CA-T-G 3’) were removed using T4 DNA polymerase. After ligation in the presence or absence of XhoI linker and transfection of JMlOl, the mutant phages M13XI7 and M13XI15 were obtained (Fig. 5). They carry, respectively, a

7-nucleotide segment and a 15-nucleotide segment (containing a XhoI site) inserted at the Hinff site. (ii) Construction of deletion mutants

the limited polymerization Using method described above, M13X9BS was cleaved selectively at the Hue11 site located in the antiterminator stem. The 3’ protruding ends (5’ G-C-G-C 3’) were digested by T4 DNA polymerase. After ligation and transfection of JMlOl, a deleted phage with the structure of M13XA2 was isolated (Fig. 5). Other deletion mutants were obtained using the Ba/31 exonuclease activity after cleavage of M13XIll at the SphI site. Unidirectional deletions with the right and left end-points locat,ed either 69 nucleotides upstream from the Hi&I site or 29, 47 and 53 nucleotides downstream from the Hinff site, respectively (M13XD9L, M13XD9R, Ml2XD2R and M13XD4R) were obtained (Fig. 2). As shown in Figure 5, the unidirectional deletion created in the mutant phage Ml3XD9L has removed a 69.nucleotide fragment containing the leader peptide sequence and its associated ribosomc binding site. The M13XD9R mutant. has lost 29 nucleotides downstream from the Hi&l sit,e comprising the 5’ half of the antiterminator stemand-loop structure. The longer deletions isolated in M13XD2R and Ml3XD4R (47 and 53 base-pairs) extend within the transcription terminator stem (RNA segment 3 in Fig. 5). It should be noted that t,hese four deletion mutants contain a IO-nucleotide sequence (the intact XhoT linker plus 2 nuclrotidrs

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. . . .. :. :. ... ... ..* .* .. : :. :. :* . : : .. .. .. - r* _.:. _ .,. _ .. :. .. * . * ... ... ... . . . .. .. ..

39

Mutagenesis of pheS, T Attenuator originating from the S$I linker) inserted at the deletion site. A last deletion mutant lacks the DNA sequence between the HinfI and HpaII restriction sites (Fig. 1). This large unidirectional deletion mutant was obtained fortuitously by Bal31 digestion in the SphI site of M13XIll and has inserted the trinucleotide A-G-G at the deletion site. In this mutant (M13XD22 in Fig. 5), all the possible secondary structures in the pheS, T regulatory region have been deleted. of point mutations (iii) Isolation To investigate in more detail the nature of the sequences involved in the attenuation mechanism expression: regulating pheS, T several point’ were constructed within the 150mutations nucleotide sequence corresponding to the leader RNA. Single-stranded DNAs of M13XSBS or MlSXSBS were hybridized to the XhoI-cut replicative form DNAs of the unidirectional delet)ions mutants described above. Heteroduplexes were then treated with sodium bisulphite (Everett. & Chambon, 1982). This mutagen selectively deaminates cytosine residues present in single-stranded DNA. Therefore. only those residues located in the single-stranded gaps of the heteroduplexes corresponding to the deletions could be modified. Depending on the cloning of the SauSA-SmaI fragment in M13mp8 or mpg, either G to A or C to T t’ransitions were obtained in the sense strand of the DNA. Ten points were selected and fully characterized by sequence analysis as described in Materia’ls and Methods (Fig. 3). Three mutants named M13XP812, 846 and 845 carry G to A transitions in the translation initiation region of the leader peptide. M13XPS43 and M13XP837 are single G to A transitions within the leader peptide

changing, respectively, the serine codon A(:C to an asparagine codon AAC and the opal stop codon UGA to an ochre codon UAA. In addition, both mutations severely affect the stabilit>y of the “protector” structure postulated by Fayat et al. (1983). As shown in Figure 5, M13XP817, P914 and P924 carry mutations in the RNA strand 2 involved in the formation of the antiterminator. The M13XP909 single mutation affects a basepaired region stabilizing the antiterminator structure (this region is removed by t.he M13XA2 deletion). Finally. the M13XP942 mutation is a C to T transition in the RNA strand 3 involved in the of both the antiterminator and formation terminator st’ructures. (b) Efj’ect of the mutations on the rqulation pheS, T operon

of the

In order to study the effect of the mutations on the regulation of the phe8, T operon, the 20 mutagenized fragments (plus t’he control wild-type fragment) were transferred in 1 phages a.s described in Materials and Methods. The structure of the resulting IX phages is shown in Figure 4. The 1X phages carry the mutant fragment joined at the SmaI site to the pheS, T-ZacZ fusion originating from phage AML2 (Springer et al., 1983). This fusion is a promoter-distal operon fusion of t,he pheS, T operon (up to the BamHI site within pheT) with the 1acZ st’ructural gene, whose own promoter has been replaced by DNA internal to the trp operon. /I-Galactosidase is expressed by the IX phages from a hybrid transcription unit made of the intact pheS, T promoter, the mutated pheS, T regulatory region. intact pheS, the 5’ end of pheT, internal trp DNA and the la& structural gene carrying its own translation

mutation

initiation

in

the

site.

The

attenuator

effect

of

region

on

each

the

Figure 5. Sequence and physiological effect of the mutations isolated in the leader region of the pheS, ‘I’ operon. The I)NA sequence corresponding to the wild-type 150-nucleotide leader RNA (Fayat et al., 1983) is represented (hyphens have been omitted for clarity). The amino acid sequence of the leader peptide is shown and its presumed ribosome binding site is underlined. The HinfI and Hue11 cleavage sites are indicated by arrows. Above the sequence, the potential pairing regions in the leader mRKA are symbolized according to Keller & Calvo (1979). The complementary sequences that form a Watson-Crick pairing region are represented by rectangles, the unpaired nucleotides forming the hairpin loop are represented by a line joining them. When a stem is formed by more than one region on one or both strands, the rectangles are joined by, dashes representing either the bases or interior loops or bulged-out bases on one strand. Hatched rectangles are pan-mg regions that are indispensable for the attenuation control mechanism. These regions are numbered 1 to 4. Open rectangles are auxiliary pairing regions that stabilize the secondary structures. Each potential structure is presented on a single line, except for the protector structure, which has been separated into protector (A) and the protector extension (B). C is the antiterminator (or pre-emptor) structure. D is the transcription terminator structure. The different mutants can be classified in 3 groups. The first group contains the deletion mutants (j.XDgT,, LXDSR. IXDZR, IXD4R, 1XA2, AXD22). The deleted nucleotides are represented by dashes between brackets; the linker nucleotides are shown between the brackets. In the case of 1XD22, the deletion ends exactly at the level of the HpaII site shown on top of Fig. 1. The second group contains the 4 insertion mutations at the Hi&I site (1.X13, IXI7, AXIll, 1X115). The 8 nucleotides surrounding the insertion point are shown. The third group contains the point mutations (LXP812 to 1XP942). Only modified nucleotides (C to T or G to A) are shown. In all cases, the unmodified bases are represented by dots. On the right part of the Figure, the relative transcription levels (calculated from Table 2) in the structural part of the pheS, T operon are indicated for each mutant in the 2 miaA + and miaA backgrounds. The transcription level of the wild-type fusion in a miaA+ strain was normalized to 1. In addition, t,he miaA - values were multiplied by a corrective factor of 350/263.7 = 1.33 (see values for 1XD22 in Table 2) which is considered to represent, under our conditions, the constant deviation between transcription levels in miaA + and miaA backgrounds (see the t,ext).

40

J.-F. Mayaux

Table 2 /I-Galactosidase levels synthesized from

YlRl and Y2Rl lysogenized with lambda phages carrying different mutations in the regulatory region of the pheS, T-1acZ fusion

Phage

YlRl

(miaA+)

IXSRS (“wild-type” fragment) IXDSL IXDSR LXDZR AXDIR IXA2 E.XD22 iXI3 1x17 AX111 AX115 1XP812 IXP846 1XP845 iXP843 iXP837 1XP817 1XP914 ZXP924 IXP909 i.XP942

53.2_+ I.9 (6) 163.2k7.4 (4) 56.1 If: 1.9 (4) 177.3k3.3 (4) 24O.Ok7.1 (4) 51.2k2.6 (4) 350.0+ 24.2 (4) 74.Ok4.3 (5) 167.2+ 10.0 (4) 288.9+ 16.4 (4) 287.1 If: 9.9 (4) 114.355.4 (6) 1 lO.O+56 (4) 80.2k4.2 (5) 73.3k3.6 (4) 72.1+ 3.8 (4) 18.4k1.9 (4) 38.2& 2.7 (4) 19.9+_ 1.0 (4) 52.4 k 2.7 (4) 946+4.8 (4)

Y2R1 (mid

-)

99.3 + 8.3 (7) 114.6_+ IO.7 (4) 51.7k4.8 (4) 131.7+f3.9 (4) 151,3+11.6 (3) 71.0+_58 (4) 263.7 + 14.6 (4) 103.9_+ 7.0 (5) 131.6k8.4 (4) 202.7 + 9.8 (4) 193.5+ 14.6 (4) 94.2 ;t IO.3 (6) 87.7 + 6.1 (6) 48.Ok5.9 (4) 115.1k5.6 (4) 111.1+3.2 (4) 10.8+1.1 (4) 84.2 * 9.3 (4) 22.4i 2.9 (4) 1120+ 6.6 (4) 123.154.8 (4)

The numbers are averages k standard deviation over 8 to 24 measurements of j?-galactosidase (units according to Miller, 1972) between 0.2 and 0.8. The number in parentheses is the at &c number of independent clones analysed in each experiment to identify monolysogens. The lysogens were grown in MOPS/ glucose medium (Neidhardt et al., 1977) for several generations at 37°C before P-galactosidase measurements were made. The doubling times of the different YlRl lysogens were 80+ 10 min: for Y2R.l lysogens, doubling times were about 5Ooj longer.

transcription of the operon is thus measured by the level of P-galactosidase activity in a strain lysogenized by one copy of the corresponding IX phage. In addition, the miaA allele dependence of fi-galactosidase synthesis was chosen to monitor the effect of the mutations on the amplitude of the attenuation control. The miaA product modifies the adenosine residue 3’ to the anticodon of several background, tRNAs such as tRNAPh’. In a miaAboth pheA (Gowrishankar & Pittard, 1982) and pheS, T operons (Springer et al., 1983) are derepressed. It is presumed that the non-modified adenosine alters the translational properties of tRNAPh’ and causes ribosome stalling at the phenylalanine codons of the leader peptides, hence derepressing both operons. The /I-galactosidase levels synthesized from monolysogenic derivatives of the E. coli strains YlRl (mia+ recA-) and Y2Rl recA-) are shown in Table 2. (miaA(i) Deletion of the alternative

mRNA

secondary

structures

As expected from previous studies (Springer et phe8, T-la& al.. 1983, 19&i), the “wild-type” fusion carried by IXSBS is derepressed in the presence of the miaA allele. In the 1.XD22 mutant’,

et al. where all the alternative secondary structures of the R’NA. including the transcription terminator of the attenuator. have been deleted, the transcription of the operon increases six- to sevenfold and the miaA-associated derepression is lost. In fact, the expression of fi-galactosidase in the 1XD22 mutant is 25 to 30% smaller in Y2Rl than in YlRl. Such a. difference is likely to reflect altered translation of /?-galactosidase in the miaAbackground. To simplify t,he interpretation, t,he miaA p-galactosidase levels were multiplied (in Fig. 5) by a factor of l-3. corresponding to the ratio of fi-galactosidase activity of J.XD22 in a miail + strain over that in a miaA- strain. In addition, all levels were normalized to the activit’y of the wildtype fusion ;IXSBS measured in the miaA’ strain. (ii) Mutations in the region C$ the leader peptide In the case of the AXDSL mutant, where the entire leader peptide region and its translational initiation signal are lost, /Cgalactosidase activity is increased threefold. As expected from the attenuation model, the antiterminator structure is free t.o form in the absence of a leader pept,ide sequence. i.e. in the absence of translating ribosomes. It should be observed that the nXD9L mutant has also lost the capability to form the protector structure (the l-2 pairing in Fig. 5): which is supposed to maintain repression in the absence of t,ranslation of the leader peptide (Fayat et al., 1983). Three mutants were shown to carry alterat,ions in the translat,ion initiation region of the leader peptide (Fig. 6(a)). The mutations are changes either in the presumed ribosome binding site (1XP812) or in the start codon of the leader peptide (1XP845 and AXP846). These mutations eliminate the ,miaA effect but cause a 1.5 to twofold derepression of the operon. These derepressions are most probably explained by the fact that the antit)erminator formation (2-3 base-pairing) is favoured. This means that, the protector format,ion is rare (structure l-2). This can be explained only if translation initiation of the leader peptide is still possible (see Discussion). Two mutations (IXP843 and 1XP837) isolated in the end of the leader peptide change two codons and affect. the stability of the protector structure (Fig. 6(c)). These mutations cause a weak but significant derepression of the operon and do not allele dependence. This the miaA modify observation suggests that t,he protector structure plays a minor role under c*ondit#ions where translation is active. Tn the case of AXP837, the CGA leader peptide termination codon is changed to UAA. As derepression is the same as with LXP843, this termination codon exchange does not seem to affect the attenua,tor response. The importance of translational coupling in the regulation of the phe8, T operon by at,tenuation is strongly suggested by the study of insert.ion mutants atet~heHinfI site. In these mut.a,nts (2x13. 17. 111 and

115).

the

distance

betweerr

the

end

of

41

Mutagenesis of pheS, T Attenuator

AX111 and 1X115 mutants even carry a stabilized protector, because the stem is lengthened by the wliaA base-pairs. However, additional dependence is lost and the derepression of the operon drastically increases as the length of the insertion increases, reaching a plateau stimulation of five- to sixfold.

(iii) ,Uutations in the antiterminator

Figure 6. Locat’ion of some mutations in the leader mRNA sequence of the pheS, T operon. The important segments 1. 2, 3 and 4 are indicated by filled bars along with the positions of G t’o A or C to U mutant changes, The leader peptide coding sequence is underlined with a boxed stop codon. For each mutant, computed free energy (AG; Rloomfield et al., 1974) of secondary structure formation is shown between parentheses. Stability of t’he corresponding wild-type structures is indicated between brackets. (a) The point mutations affecting the translation initiation region and the terminator structure are shown. The insertion site of mutants X13. X15, XI11 and XI15 is symbolized by an arrow. The presumed Shine & Dalgarno (1974) sequence A-G-G-A is underlined and the alternative signal UAA is indicated by dots. (b) Mutations affecting the stability of the antiterminator structure are shown and the tetranucleotide deleted (1XA2) in the antiterminator extension is indicated by a hatched box. (c) The 2 pointmutations isolated in the protector sequence are shown. (d) The pseudo-antiterminator structure stabilized by the base-pairing between strand 3 and the XhoI linker inserted at the deletion site of 1XD9R is represented. For clarity, sequence hyphens have been omitted and basepairing is indicated by dashes.

is the leader peptide and the antiterminator gradually increased by 3, 7, 11 and 15 nucleotides (Fig. 6(a)). Moreover, these insertions are located in the loop of the protector structure and should not, affect the stabilit’y of the l-2 base-pairing. The

structuw

The next group of mutations includes both single and multiple changes within different parts of the structure (Fig. 6(b)). These antiterminator mutations have a down phenotype even in the miaA+ background (see AXP817, P914 and P924 in Fig. 5). The effects of these mutations on downstream transcription correlate satisfactorily with their effects on the predicted stability of the antiterminator. The comparison of the triple mutants 1XP817 and AXP924 provides a strong evidence that strand 2 is responsible for the competition with the 3-4 base-pairing of the terminator. In both mutants, the stability of the whole antiterminator stem is almost equally affected (from -24.5 to 5.2 and -6.6 kcal/mol, respectively: 1 cal = 4.184 J) even if strand 2 is altered much more in IXP817 than in AXP924. The derepression caused by the miaA significant with IXP924 and allele is still abolished with Our completely 1XP817. interpretation is that the RNA strand 2 of IXP924 can still base-pair with RNA strand 3 (see Fig. 6(b)) and thus retains the capability to compete weakly with the formation of the terminator structure (3-4 base-pairing). On the contrary, in the case of 1”XP817, where the miaA allele effect is totally lost, the 2-3 base-pairing is completely abolished. Mutations (AXA2, IP909) isolated in the part of the ant’iterminator stem that does not base-pair with RNA strand 3 (antiterminator extension; see Fig. 6(b)) have a moderate of a negligible effect on pheS, T regulation. These results demonstrate that the antiterminator extension stabilizes the antit’erminator structure but does not play an essential role in the attenuation mechanism, as predicted from t’he model proposed by Fayat et al. (1983). The 29-nucleotide deletion characterized in iXD9R eliminates a part of the sequence corresponding to the antiterminator structure. However, the P-galactosidase level is not decreased in the miaA+ background, as in the case of the point mutations described above (P817, P914 and P924). This can be explained bv the possibility of creating a pseudo-antiterminator structure stabilized by the base-pairing between the XhoI linker inserted at the deletion site and strand 3 of the terminator (Fig. 6(d)). This pairing might still cause a weak coupling between translation of the leader peptide and the formation of the terminator. similar to the mechanism postulated for the native pheS, T control region. This weak coupling could explain the miaA allele effect observed in this case.

42

J.-F. Mayaux et al.

(iv) Mutations in the terminator structure In mutants AXDBR and lXD4R, read-through transcription increases three- to fivefold, while the miaA dependence is lost. These mutants have lost the antiterminator control region as in mutant AXDSR, but, the right end-points of the deletions now reach the RNA segment 3. It can be concluded; therefore, that an intact, terminator stem is required to repress the operon. A unique C to T t,ransition in the end of strand 3 was isolated also (1XP942 in Fig. 6(a)). This mutation decreases the computed stability of the terminator structure from - 17.6 to - 13.8 kcal/mol and causes a nearly twofold increase of the expression of the operon in the mlaA f strain. In addition, the derepression is smaller in the miaA background, probably because the mutation also affects the stability of t,he antiterminator ( - 19.7 instead of - 24.5 kcal/mol).

4. Discussion The techniques for site-directed mutagenesis of DNA in vitro have been introduced recently (reviewed by Shortle et al., 1981). Deletions, insertions or point mutations can be created with remarkably high frequencies at almost any location in a cloned DNA. The use of Ml3 sequencing vectors carrying multi-cloning sites (Messing, 1983) extends the ease and versatility of these techniques. For instance, as shown in this work, the singlestranded viral form of a recombinant phage can be made partially double-stranded in a defined region, t)hus allowing that only the restriction sites of the double-st#randed fragment should be recognized and cleaved by the corresponding restriction enzymes. On the ot’her hand, base transitions ran be introduced into the single-stranded gaps of specific heteroduplexes by treat,ment’ with bisulphite (Everett, & Chambon, 1982). These techniques are well-suited to probe the functions of the different segments composing the pheS, T attenuator region because (1) in spite of the mild conditions of mutagenesis, more than 75% of the sequenced clones are mutants. (2) each mutant can be sequenced rapidly before any physiological study. Therefore, potentially silent mutations are not overlooked. The effect of the mutations on transcriptional attenuation was measured after introducing each mutation into the chromosome. in single-copy form, using a 1 recombinant phage carrying a pheS, Tlad fusion. The properties of strains bearing definite mutations agree well with the predictions of the attenuation model (Fayat el al., 1983) and give a better insight into the pheS, T regulatory process. The large deletion D22 has removed all potential secondary RNA including struct’ures the transcription t,erminator. Tt causes a six- to sevenfold increase of the expression of the operon. We can thus estimate that, in wild-type bacteria growing at 37°C in glucose minimal medium, only

150/o of the Rh’A polymerase molecules that initiatr transcription at the phd, T promoter read-through the entire operon. The maximal efficiency of t,he t,erminator is suggested by mutant P817. This mutant ca,rries a triple mutation in RKA strand 2’. which deeply affects the stability of thr Z--3 antiterminator structure. As the mutated structure cannot compete significantly with the formation of the terminator 3-4 base-pairing, the m&A effect disappears and only the bass1 read-through level of the terminator is measured. Following the terminology proposed by Sbroynowski et nl. (1982). the P817 mutations cause superat’tenuation: that is. increased and uncontrolled termination a.t the a,ttenuator. Since the basal level of transcript.ion is about one-third of the wild-type level, we conclude t’hat att,enuation might permit regulation of’ expression of t’he ph.&“. T operon over a maximal 20.fold range. The P817 and I’924 mutations also suggest, tha,t when neitJher I-2 nor 2-3 can form. structure 3-4 is sufficient. for termination. Studies of the deletion mutant.s D2R and D4R show that an int.ac*t segment 3 is necessary for a maximal terminatiolh. This is indicated also by P942, which cxarries a single point-mut,at)ion at the end of segment 4. Ttr previous experimenbs (Springer et al.. 1985). it was shown using gradua,l delet)ions generated from the SmaI sife (Fig. I) that the stretch of ‘l’ residues (nucleotides 142 to 148 in Fig. 5) was also part of t,he terminator. The combination of this result and of our present data allow us t.o precisely map ir/ /ail the boundaries of the termination signal of the pheS. T at,tenuator. This mapping confirms the result of transcription experiment,s in r:ifro (Fayat, pt al.. 1983). The role of the antiterminator as a competitjor of the terminat,or is demonstrated by the deletion mutant D9I,. Since the delet.ion removes the wholr leader peptide and the 5’ half of the protector structure. the formation of t’he ant’iterminator is favoured on the nascent RNA, which is reflected b? the threefold derepression of the operon. Multiple or single mutants with alterat(ions in the transla.tion initiat,ion region of the leader peptidr are slightly derepressed ( 62-fold) and have lost t.he ,miad allele dependence. These mut,ations strong]!suggest that translation of the pheS1 leader pept.ide is involved in the attenuation cont.rol of phuR, 7’ expression. Nevertheless. the ph.eS, 7’ at,tenuator differs from the trp a,ttenuator. since modifying the st’art codon for the frp leader pept,ide causes reduced expression of the trp operon (Kolter & Yanofsky, 1984). r\ possible explanation is t’hat. in t.his study. t,he mutations do notf totally prtbveni t II~’ binding of’ ribosomes ;tn
Mutugenesis

of pheS, T Attenuator

to A-G-A-U, the trinucleotide (:-,4-U could still base-pair with C-C-A of the 3’ end of the 16 S ribosomal RNA (3’ A-U-C’-~-C-C-C-C-ARC-U-A-G). In addition, it is quite possible that on both mutant and wild type the U-A-A sequence just after the X-G-G-V or the A-G-A-C sequences could he used as an ahernative ribosomal binding site. In the caase of P845 and P846. where the AUG initiator codon is changed to AUB: we are obliged to suppose that translat,ion initiation could still take place even if the rate is decreased severely. Assuming that translation of the leader peptide occurs on the three mutants, we shall now discuss t.he effect of the t.ranslation initiation efficiency on the attenuator response of pheS, T and trp operons. Under non-starvation conditions, according t)o the model proposed by Manabe (1981), the readthrough probability is sensitive to the rate of translation initiation of the leader peptide. Three general cases can be considered: (1) the rat,e of initiation is high. Tn this condition, the translating ribosome reaches the stop codon of the leader with high pept.ide fa,st enough to prevent probability the antiterminator formation on the nascent leader mR,NA: transcription terminates at the t,erminator. (2) The rate of initiation is ver? low. In this case, the probability of hindering the protector formation by a translat,ing ribosome is small and transcription t’ermination is also favoured. (3) The initiation rate is intermediat,e. Tn this case, the read-through probability can reach a maximum value. Tn other words, read-through probability first increases, reaches a maximum and then decreases as translat,ion initiation rate of the leader peptide increases. When the wild type translation initiation rate of a given leader peptlde is slightly superior to the value that gives the maximum

reatl-through

probability.

any mutation

decreasing t,his rate is going to decrease reatlthrough. This could be the case of the trp operon. On the contrary, when the wild type initiation rate is superior to the value ensuring the maximum read-through. mutations decreasing this rate can increase the read-through probabilit’y. This could be the case of pheS. T operon. In other words. the different response to alterations of leader peptide init,iation regions observed in the case of trp and p//es, 7’ operons could be explained in t,erms of different t,ranslation init,iation efficiencies of the wild type leader peptides. tt1r insert)ion mutations Finally, provide of the main suggestive evidence in support prediction of the attenuation model, i.e. that the format’ion of t’he 2-3 antiterminator structure is regulated b\- the translation of the leader pepMe. In these mutants. an almost maximal derepression of the operon is observed when the leader peptide is shift’ed 11 nucleotides away from the antit,ermina.tor. As a likely explanation for this derepression. we propose that a ribosome located on t,hr last c&ons of the leader peptide becomes unable t’o prevent the formation of the antiterminator structure by steric hindrance. as the

43

distance increases between the leader peptide stop codon and the RKA segment 2. In other words. the insertions uncouple the formation of the 1-3 RXA structure from t’he translation of the leader peptide. Strikingly, the maximum derepression is reached for an insertion length (between 7 and 11 nucleotides) corresponding to the distance between the last phenylalanine codon and the stop codon of the leader peptide. This suggests that the location of the last’ phenylalanine codons is adapted to prevent a stalling ribosome from hindering the formation of t#he antiterminator structure. We thank Y. Mechulam and P. Mellot, for preparing in vifro packaging extracts. This work was supported in part by the following grants, Centre National de la Recherche Scientifique (L.A. no. 240). Ministere de I’Industrie et de la Recherche (dkcision d’aide no. 83VO623). J.-F.M. was a from Rhbne-Poulenc recipient, of a studentship Recherche.

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Manabe. T. (1981). J. Theo& Biol. 91, 527-544. Maniat,is, T.. Fritsch. E. F. & Sambrook, J. (1982). Molecular Cloning, Cold Spring Harbor Laborat,ory Press, Cold Spring Harbor. Mayaux, J. F.. Springer, M.. Fromant, M.. Graffe, M. & Fayat, G. (1984). Gene 30, 1377146. lIessing, J. (1979). Recomb. DNA Tech. Bull. 2. 43-44. Messing, J. (1983). In Methods in Enzymology (Wu, R.. Grossman, L. & Moldave, K., eds), vol. 101, part C, pp. 2928, Academic Press, New York. Messing, J. & Vieira, J. (1982). Gene, 19, 269-276. Miller. J. H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. Neidhardt: F. C.. Bloch, P. L., Pedersen. S. & Reeh, S. (1977). J. Bacterial. 129; 378-387. Plumbridge, J. A. & Springer, M. (1980). J. &fol. Biol. 144, 595-600. Plumbridge, J. 4., Springer, M., Graffe, M.. Grousot, R. & Grunberg-Manago, M. (1980). Gene, 11. 33-42. Sanger, F., Coulson. A. R., Barrel, B. G., Smith. A. J. H. 8: Roe, B. A. (1980). J. Mol. Biol. 143. 161-178. Shine. J. & Dalgarno, 1,. (1974). Proc. Xut. Acad. Sci., U.S.A. 71. 3342-1346. Edited

et al. Shortle, I>. & Rotstein, D. (1983). In Methods in Enzymology (Wu. R.; Grossman, L. & Moldavr. K.. eds), vol. 100, pp. 457-468, Academic Press, New York. Shortle. D., DiMaio. D. & Nathans, D. (1981). Annw. Ret>. Genet. 15, 265-294. Springer. M.. Graffe: M. & Hennecke, H. (1977). Proc. Nat. Acad. Sci., 1,T.S.A. 74, 3970-3974. Springer, M.. Trudel, M.. Graffe, M.. Plumbridge. J. A., Fayat. G.. Mayaux, ,J. F.. Sacerdot. C.. Blanquet. 8. 8t Grunberg-Manago. M. (1983). J. idol. Biol. 171. 263-279. Springer. M.. Mayaux. J. F.. Fayat, G.. Plumbridge,
by P. Chumbow