Cotranscription of Two Genes Necessary for Ribosomal Protein Lii Methylation (prmA) and Pantothenate Transport (panF) in Escherichia coli K-12 Downloaded from

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Protein Lii Methylation (prmA) and Pantothenate

Transport (panF) in Escherichia coli K-12 Downloaded

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Anne Vanet, Jacqueline Plumbridge, Jean-Herve Alix

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Anne Vanet, Jacqueline Plumbridge, Jean-Herve Alix. Cotranscription of Two Genes Necessary for Ribosomal Protein Lii Methylation (prmA) and Pantothenate Transport (panF) in Escherichia coli K-12 Downloaded from. Journal of Bacteriology, American Society for Microbiology, 1993, 175 (22), pp.7178 - 7188. �10.1128/jb.175.22.7178-7188.1993�. �hal-01898765�

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Vol. 175,No.22 JOURNALOFBACTERIOLOGY, Nov.1993, p.7178-7188

0021-9193/93/227178-11$02.00/0

Cotranscription

of

Two

Genes

Necessary

for

Ribosomal

Protein

Lii

Methylation

(prmA) and

Pantothenate Transport

(panF)

in

Escherichia coli

K-12

ANNEVANET,* JACQUELINEA. PLUMBRIDGE, ANDJEAN-HERVE ALIX

Institut de BiologiePhysico-Chimique, (URA 1139), Centre National de la RechercheScientifique,

13, ruePierreetMarie Curie, 75005 Paris, France

Received 1 June1993/Accepted 17 September1993

Genetic complementation andenzymeassayshave shown thatthe DNAregion betweenpanF,whichencodes pantothenatepermease,and

orfl,

thefirstgeneofthefisoperon,encodesprmA,thegenetic determinantforthe

ribosomal protein Lll methyltransferase. Sequencing ofthis region identified one long openreading frame thatencodesaprotein of31,830 Da and correspondstotheprmAgene.Wefound,both in vivo and invitro, that prmAis expressed frompromoterslocatedupstreamofpanF and thus that the panFandprmAgenesconstitute

abifunctionaloperon.Welocated the major 3' end ofprmA transcripts 90 nucleotides downstreamofthestop codon of prmA in the DNA region upstream of thefis operon, a region implicated in the control of the expression ofthefis operon.Although nopromoteractivity wasdetected immediately upstream ofprmA, Si

mapping detected5' ends of mRNA in this region, implying that somemRNAprocessing occurswithin the bicistronicpanF-prmA mRNA.

Posttranslational modification of several ribosomal proteins is a general phenomenon observed in both prokaryotes and eukaryotes (2). Although much information ontheexpression of the genes for bacterial rRNAs and ribosomal proteins is available(29), little attention has been giventotheir

posttrans-lational modifications, which are actually quite numerous. In

Escherichia coli, proteins suchasS5, S18, L12, andEF-Tu are

acetylated at their N-terminal residues (L7 is the acetylated form ofL12). Others modifications include addition ofamino

acids to the polypeptide chain, e.g., addition ofone to four glutamic acid residues to the C terminal ofribosomal protein

S6 (25, 30). However, the most frequent modification is methylation. Several ribosomal proteins, e.g.,

Sli,

L3, L7/L12,

Lii, L16, and L33, as well as EF-Tu and IF3, have

N-methylated amino acids at specific positions (reviewed in reference 2). Lii is the most heavily methylated ribosomal protein, with three trimethylated amino acid residues: two

Ns-trimethyllysines at positions 3 and 39 (19) and an amino-terminalNox-trimethylalanine (18, 35). Methylation of

riboso-mal proteinL1I requiresS-adenosyl-L-methionine as amethyl

group donor(4) in a co- or posttranslational event.

Mutations calledprmA, which result in an unmethylated form of

L1i,

have beenisolatedindifferent laboratoriesandby

independent procedures (15-17, 27). The prmAl locus was

mapped by Colson et al. (14) to 71 min on the E. coli chromosome. This methylation might be expectedtoaffect the function of L1I. However, theprmA mutant strains were all

phenotypically indiscernible from their wild-type parents,and

therefore the function, if any, of this energetically costly

methylation of protein

L1i

is unknown.

L1i

has been

impli-cated in several aspects of ribosome function and assembly, namely, the stringent response in vivo (6, 43-45) and invitro,

ribosomal subunit association (21,31, 40), thebinding domain

of the antibiotic thiostrepton (60), ribosomal protein L16

assembly during 50S subunit reconstitution (9, 23), protein

*Correspondingauthor.

synthesis

termination (7, 58, 59), and the ribosomal GTPase

domain

(20,

48). Despite all of these suggestive results, the

precise role for Lii in the ribosome is obscure.

Moreover,

mutantsapparently lacking Lii areviable(56,

57).

The L1I molecules isolated from prmA strains are severely undermethylated. In vitro methylation experimentswith

ribo-somes from theprmA strains and with extracts fromprmA+

bacteria and radioactively labeled

S-adenosyl-L-methionine

show that theprmA mutations result in loss ofmethyl groups

from both the N-terminal alanine and the internal lysine

residues

(5, 14,

15). However,someresidualLii

methyltrans-feraseactivitycanbedetected inextractsfrom strainscarrying prmAl orprmA3mutations(2,15).This residualactivitycould

accountfor thelack ofaphenotype associatedwith theprmA

mutations. Since single mutations apparently result in the

absence of both internal and N-terminal methylgroups from

Li1,itseemspossible that thesame enzymeisresponsiblefor

themethylationof allthreeamino acids. If this is the case, then the distinctive trimethylation of two different amino acids at threedifferentloci when all of the otherlysine residuesof the

protein are unmodified poses an interesting problem of en-zymespecificity. We cannot, however,rule out thepossibility

thattwoormoreenzymes are necessaryandthatprmA isthe

genetic determinant of thelimitingstepwhich prevents subse-quentmethylations.

Tostudythe role ofmethylation onLii function,we have

undertaken a systematic study of the prmA gene.

Chromo-somalDNAfragmentscarrying theprmAgene wereisolatedin a series of X transducing phages by complementation of a

thermosensitive mutation in the nearby genefabE (now re-named accB; encodes the biotin carboxyl carrier protein, a subunit ofacetylcoenzymeAcarboxylase)(3).ThepanFgene

(encodes pantothenate permease) has been mapped to the

samechromosome region (62).All of the accB+ Xtransducing

phages which carried panF were also found to be prmiA+,

showingthat genes prmnA andpanF arecloselylinked(3).The nucleotide sequence of panF has been determined (28). We presentheretheDNAsequenceof prmA and ananalysisofits

transcription. 7178

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TABLE 1. Bacterialstrains, plasmids, and phages used in this study

Strain,plasmid, Genotype Referenceororigin

orphage

E.coli strains

IBPC5321 thi-1argG6 argE3 his-4xyl-5 mtl-l tsx-29 rpsL AlacX74 46

MB984 srl::TnJO recA prmA3 IBPC101(47) x MB1984(3)

byP1 transduction

JM109 recAI endAIgyrA96 thihsdRI7supE44relAI A-A(lacproAB)F' (traD36proABlacIq 65

lacZAM15)

MB1541 thr-I leu-6 trp his-4 argHlthyAl thi-1xyl-7 tonA2 supE44rpsL9ficTs' prmA3 3

Plasmids

pBR322 amp tet 10

pFA accBC panF amp 3

pAF2 panFprmAorfl amp Thiswork

pAF3 panFprmA amp This work

pRS415 amp 'lacZ 54

pAO1 panFprmA-lacZamp This work

pAO2 panF-lacZamp This work

pROl 'panFprmA-lacZamp This work

pRO2 'panFprmA-lacZamp This work

pUC(PrmA) placprmA This work

Phages

X6G3 accBC panFprmA orflfis 33

XFA6 accBCpanFprmA 3

XRS45 'lacZYA 54

XRS/A01 panFprmA-lacZ This work

XRS/A02 panF-lacZ This work

XRS/RO1 'panFprmA-lacZ This work

XRS/RO2 'panFprmA-lacZ Thiswork

a Aprime denotes thatthegeneistruncatedonthat sidesothat thepromoter regionisabsent.

MATERIALS AND METHODS

Strains and media. Thebacterial strains andplasmids used

in this study are listed in Table 1. All strainswere routinely

grown in Luria-Bertani (LB) medium. Synthetic MOPS

me-dium(41) containing0.2%glucose and 50jigeach of arginine

andhistidinepermlat30°Cwasusedinbacterial cultures for

measurements of P-galactosidase activities as previously

de-scribed (39). Plasmidswere propagated inE. coli JM109 or

IBPC5321. X6G3 is from the E. coli library of Kohara etal. (33). Plasmid pUC(PrmA) carries the prnzA structural gene,

between oligonucleotides CHIEN and IFRO (see Fig. 2),

cloned downstream ofthe lac promoter. Itsconstruction will

bedetailed elsewhere (64).

DNAmanipulationsandsequencing. Restriction

endonucle-asesand others enzymeswereobtained commercially. Radio-chemicalswerepurchasedfrom Amersham. Large-scale

prep-arations of plasmid DNA, digestions, and cloning were

performed by standard procedures (50). DNA restriction fragments and polymerase chain reaction (PCR)-generated

fragmentswerepurifiedfromagarosegels bythe freezephenol method(53)orwithGene Clean(Bio 101). ThePCR(50 ,ul)

contained 20pmolofeacholigonucleotide, 10ngofaplasmid

[pAO2, pUC(PrmA), or pAF3] template, 2 U of Taq

poly-merase, 0.2 mM each deoxynucleoside triphosphate, 10 mM

Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, and 0.01%

gelatin.PCRsweregenerallycarriedoutbythefollowingthree

steps repeated 20 times: denaturation at 92°C for 1.5 min, annealingat55°Cfor 1.5min,andextensionat72°Cfor2min. Double-stranded DNA of recombinant plasmid pAF3 was

directly sequenced on both strands as previously described

(51). For the oligonucleotides used as sequencing primers

(three foreachstrand), see Fig. 2. Dideoxy-sequencing

reac-tions using T7 DNApolymerase and a-35S-dATP were

ana-lyzed

on 0.4-mm-thick 8% acrylamide gels containing 7 M

urea.

mRNA preparation. Total RNAwas prepared from strain

JM109 or IBPC5321 grown in LB medium. Strains carrying plasmidpAF3 or pFA were grown in LB containing500 ,ug of

ampicillin per ml. Cells were harvested at an A650 of 0.7, washed with 10

mM

MgCl2, andresuspendedin 20 mMsodium acetate (pH

4.7)-1%

sodium dodecyl sulfate, and RNA was

extracted bythe hotphenolmethod (49).

S1 nucleasemapping. Tomapthe 5' ends ofpanF

andprmA

transcripts,probes uniquelylabeledatone5'endwere synthe-sized by PCR. A 20-pmol sample of one

oligonucleotide

(NAPCO

for panF and ARTforprmA)was5'end labeled with

[y-32P]ATP

(specific activity, 3,000

Ci/mmol)

and

polynucle-otidekinase. The second

oligonucleotide

was complementary

toplasmidvectorsequences upstreamof the

panFprmA

region

inserted in theplasmidusedasthetemplatefor the PCR.The reverse sequencing primer (REV17) was used with

plasmid

pUC(PrmA)

for the

prnmA

probe (see Fig.

6)

andan

oligonu-cleotidehomologoustothe amp gene ofpRS415

(RBP22)

with

pAO2for panF (see Fig. 4). The

SI

probes thus

synthesized

carried a nonhybridizing DNA extension at the unlabeled

oligonucleotide

end which

permits

differentiation between

readthrough transcription andreannealing of the probe. The labeledfragmentswereseparated from

unpolymerized

oligo-nucleotides on a 1% agarose gel and

purified by

the freeze

phenolmethod (53).

To mapthe 3' ends of

prmA

transcripts, aDNA

fragment,

RUE-IFRO (see Fig. 2), was

synthesized

by PCR. This

frag-ment was digested by

HindIll.

TheHindlIl-IFRO

fragment

waspurifiedfroma1% agarose

gel

with GeneClean

(Bio

101),

and the

Hindlll

endwas labeled with

[ot-32P]dATP

with the Klenow fragment of DNApolymerasein the presence of 0.2

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7180 VANET ET AL.

mMeach

dCTP,

dGTP, and dTTP. This 3' end-labeledDNA

fragment

was purified by elution from a 5% polyacrylamide

gel.

The end-labeled DNA probes, approximately 50,000 cpm

per

hybridization experiment,

wereincubated with 10to30p.g

ofE. coli total RNA in 80% formamide-40 mM

piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES;

pH

6.4)-I

mM

EDTA-400 mM NaCl

(final

volume,

50

Rl).

After denatur-ation at

85°C

for 10 min, hybridization was carried out

overnight

at

56°C

(5'-end

mRNA

mapping)

or 52°C (3'-end

mRNA

mapping)

and stopped by

transferring

the hybridized

mixtureto 0.4 mlof coldSI nuclease

digestion

buffer

(30

mM

sodium acetate

[pH 4.6],

250 mM NaCl, 1 mM ZnSO4, 5%

glycerol) containing

100 UofSI nuclease

(Boehringer GmbH,

Mannheim, Germany).

The

samples

were incubated at 37°C

for 45 min

(5'-end mapping)

or at

25°C

for 1.5 h (3'-end

mapping).

SI nuclease-resistant DNA

fragments

were

ana-lyzed

on

polyacrylamide

gels 1mm thickcontaining7 Murea.

The

gels

were

fixed, dried,

andanalyzedwitha

Phospholmager

(Molecular Dynamics),

which permitted quantitation of

S1-protected

bands,

or

autoradiographed

with Cronex Hi-Plus

amplifying

screens.

Reversetranscription.

Synthetic

oligonucleotides

wereused

for

primer

extension

experiments

with avian myeloblastosis

virusreverse

transcriptase

(Boehringer).

The oligonucleotides

were 5' end labeled with

[_y-32P]ATP

and

polynucleotide

kinase and separated from the labeled nucleotide

by

electro-phoresis

on

denaturing

20%

polyacrylamide gels. Typically,

15

or 30 jig ofE. coli total RNA and 0.5 pmol of the labeled

oligonucleotide

wereannealed inavolume of5

Rl.

Thereverse

transcription

reaction was

performed

as

previously

described

(61).

The

primer

extension

products

were

analyzed

on

dena-turing polyacrylamide gels

as described above.

In vitro transcription.

Templates

were made

by

PCRwith suitable

oligonucleotides.

The reaction mixture

(20

il)

con-tained40 mMTris-HCl

(pH

8.0),

10 mM

MgCl2,

100 mM

KCl,

1 mM

EDTA,

100

pLg

ofbovine serumalbuminper

ml,

50ng

of

template

DNA

(e.g.,

theAGFIN-NAPCO

PCR-generated

fragment

for

mapping

of thepanF

promoter),

and0.3to 1 Uof

theE. coli RNA

polymerase

holoenzyme

(Boehringer).

After

15min of

preincubation

at

37°C,

a

transcription

wasinitiated

by

additionof 400 FMeach

ATP, CTP,

and GTP and 100 FM

[0-32P]UTP.

After 15 min, the

samples

were

precipitated

with ethanol and

analyzed

on

denaturing polyacrylamide

gels as

described above.

Construction of

panF-lacZ, prnzA-lacZ,

andpanFprmA-lacZ

transcriptional

fusions. All fusionswere constructedon

plas-mids in vitro and transferred to X

by

in vivo recombination. PlasmidpAOI

(panF

prmA-lacZ)

wasconstructedby

inserting

the 2.4-kb BamHI

fragment

from

pAF3, carrying

a small

fragment

of

pBR322,

the end of accC

(fabG),

the

entirepanF

gene,and thefirst 10nucleotides of

prmA,

into theBamHI site

of

pRS415

(54).

Plasmid pAO2

(panF-lacZ)

was constructed

similarly,

by

inserting

the 1.95-kb

BamHI-BglII

fragmentfrom

pAF3

(the BglII

site is locatedatthe end ofpanF[see Fig. 1])

into the BamHI site ofpRS415. Plasmid

pROl

was derived from

pAOI

by

deletion ofthe DNAbetween the EcoRIand

BglII (at

theend ofpanF)sites(see Fig. 1).Plasmid pRO2was

made

by

inserting

the 1,530-bp

EcoRV-to-BamHI

fragment

from pAF3 into pRS415 digested by

SmaI

and

BamHI.

The

fusions carried

by

pAOI,

pAO2, pROI,

and pRO2 were

transferredtoXinvivo

by using XRS45

as

previously

described

(54)

to

give

XRS/AO1, XRS/AO2, XRS/ROI,

and

XRS/RO2.

Two

independent

plasmid

constructs for each fusion were transferredto

XRS45,

and two recombinants from each

plas-mid were

picked, purified,

and used to make lysates to

lysogenize

strain IBPC5321.

Monolysogens

were determined

and

,3-galactosidase

activities were measured as

previously

described

(39).

Lll

methyltransferase

activity

measurements. Ribosomal

protein

L

1I methyltransferase

activity

wasmeasured

basically

as

previously

described

(15).

To measure the

specific

Lii

methyltransferase

activity

ofa

particular

crudeextract,

bacte-riafroman

overnight

culture ofE.colicells in LBmedium with

500 jig of

ampicillin

per ml

(for

plasmid-carrying strains)were

pelleted, resuspended

in an

appropriate

volume of a buffer

containing

10mMTris-HCl

(pH 7.6),

10 mM

MgCL2,

60 mM

NH4Cl,

and 6 mM

f-mercaptoethanol,

andbroken

by

sonica-tion.The

25-jl

methylation

assaycontained 1

A260

unit of50S ribosomalsubunits ofE.coli MB1541

(prmA3)

asthesubstrate

(methyl

group acceptor), 25 jiM

S-adenosyl-L-[methyl-3H]-methionine

(3 Ci/mmol; Amersham)

as the methyl group

donor

(a

concentration

eightfold

over the reported Km

[3.2

,uM]

of the enzyme for S-adenosyl-L-methionine)

(13),

and various amounts of the bacterial extracts as a source of Li1 methyltransferase. Reaction mixtures were usually incubated at 30°C for 1 to 30

min,

and the hot 5% trichloroacetic acid-insoluble

radioactivity

was collected on Millipore filters and counted. Proteinconcentrations in the crudeextractwere

estimated

(ii),

andthefinal resultwasexpressedin

picomoles

of

methyl

groups

incorporated

per minute of incubation at

30°C per

milligram

of

protein

in the crude extract. RESULTS

Subcloning

and identificationof

prmA

asthegenetic

deter-minant for L 11methyltransferase.TheprinAgenewasfound

to be

closely

linked tothe accB (formerly fabE; encodes the

biotin

carboxyl

carrier protein subunit ofacetyl-coenzyme A

carboxylase) and

panF

(encodes pantothenate

permease)

genes at 71 min onthe E. coli chromosome (3). Of the 75 X

bacteriophages

isolated as complementing a thermosensitive

mutation inaccB, the 58 thatwerepanF were alsoprmiA

+,

showing thatpanF andprmiA are likely to be adjacent. The gene

fis,

which encodes the factor for inversion stimulation

(Fis),has beenmappedto the sameregion (32).

Comparison

oftherestriction maps of the

accB,panF,prmA,

andfis

regions

showed that these fourgenesarecarriedon Kohara

bacterio-phage

X6G3

(33) (Fig. 1).

The accB, accC (formerly

fabG;

encodes thebiotin

carboxylase

subunit ofacetyl coenzymeA

carboxylase),andpanFgeneshave beensequenced(3, 28,

34,

38),

as have fis (32) and a gene (orfl) with an unknown function positioned immediately upstream offis and

tran-scribed from the same promoter (8, 42). Located between

panF and orfi is a region of about 1.0 kb which was not

sequenced

(Fig. 1).

This region is alsopresent on the accB+

panF+ prmiA+transducingphagesof Alix(3),while it isabsent

from plasmid pFA, which complements the accB and

panF

mutationsbutnot aprmnA mutation (3). Thus, theprmnA gene should lie within theunsequenced DNA

region.

Wetherefore cloneda4.2-kbPvuIIfragment,

encompassing

thisregionfrom X6G3into theEcoRVsite ofpBR322to

give

plasmidpAF2.This fragmentcarries the end(last 136

codons)

of the accC gene, panF, and the orfi gene, as well as the

unsequenced DNA. A slightly smaller plasmid, pAF3, was

madeby deleting 1,060 bp which includedorfi (Fig. 1). This

plasmid was introduced into the prmA3 mutant strain (MB984),andLII methyltransferase activitywasmeasuredon crude extracts. MB984 (prmA3) showed about 2.5% of the

activity ofwild-type E. coli IBPC5321 (Table 2). Introduction

ofplasmid pAF3 ledto aconsiderable increase ofLii

meth-yltransferase activity, showing that plasmid pAF3

comple-J. BACTERIOL.

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B

XFA6 -\. v//

B B

accBC p&nF pnnA r

X6G3 xY

Z//

// ,xx x xy

/ccBC ~~~~p&nFprmAorf t

BEV/P EV B

tet' 'accC panF prmA or11 amp

B EV/P EV Bg B H E

1-k,V

:

tet' 'accC panF prmA amp

ESB EV/P EV Bg B

amp accC panF,prmA-lacZYA tet

ESB EV/P amp 'accC amp EV Bg panF-lacZYA tet E/Bg B

amp 'panF, prmA-lacZYA tet

E Bg B

li

X>tIj

'panF, prmA-lacZYA tet

1kb

oligonucleotides

'accC panF prmA orfl'

_GF

_

_I

- -_-

_F

AGFIN BGFIN FNAP NAPCO CHIEN AMFIP VERSE ART RUE AL IFRO

FIG. 1. Subcloning of the panF prmA region of theE.coli chromosome.RestrictionmapsofXFA6andX6G3are shownwith thelocations of

theaccBC,panF,andprmA genes.The4.2-kbPvuIIfragment of X6G3wascloned intotheEcoRV siteof pBR322togive pAF2.Deletion of the

NcoI-EcoRI fragment(by fillinginof thesiteswith the Klenowenzyme and ligation,which recreatesthe EcoRIsite)gavepAF3.The different

DNAfragments insertedinto pRS415toproduceoperonfusions with lacZ, pAO1,pAO2, pROl, and pRO2areshown. _,prmA; Eli,panF;

=,othergenes; ,lacZ; VA, phageX; II1,plasmidvector.Abbreviations:B,BamHI;P,PvuIl; EV, EcoRV; Bg,BglII;E,EcoRI;H,HindIII;

N, NcoI; S, SmaI; E/Bg, hybridsite fromEcoRI and BglII;amp,ampicillin resistance gene; tet, tetracycline resistance gene. <-*, 6-kb BamHI

fragment of XFA6 inserted into the BamHIsiteofpBR322togiveplasmid pFA (3).

TABLE 2. Specificactivities ofL1i methyltransferasein

plasmid-carryingstrains

prmAalleleon Enzymeactivity"

chromosome Specific Relative

MB984(pBR322) prmA3 0.01 0.02

MB984(pAF3) prmA3 6.04 13.72

IBPC5321(pBR322) prmA+ 0.44 1.00

IBPC5321(pAF3) prmA+ 7.34 16.68

'Specificactivitiesareexpressedinpicomolesofmethylgroupsincorporated

permilligramofproteinin thebacterialextractperminuteat30°C.

mented theprmA3 mutation (Table 2, line 2). PlasmidpAF3

gave a similar level ofLII methyltransferase activity in

wild-type strain IBPC5321, resulting in about 15-fold overproduc-tioncomparedwith thesamestraincarryingpBR322 (Table 2,

lines3 and4). This level ofoverproduction, comparabletothe

pBR322copynumber, is consistentwith the idea that theprmA locus encodes the structural genefor LII methyltransferase.

Sequenceof the prmA gene. The DNA sequence of pAF3

betweenpanF and orfi was determined (Fig. 2). One long

open reading frame (ORF) was found in the region

corre-spondingtotheprmA gene.The first ATG codonof this ORF islocated 11 nucleotidesdownstream of the terminationcodon

ofpanF.TheORFterminatesataTAA codon328 nucleotides

upstream of the orfi gene. The prmA gene codes for a

polypeptide of 293 amino acids, giving a protein with a

pAF3

pAO1

pAO2

pROl

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7182 VANET ET AL.

1

'panF CHIEN

GAATATTCAGTACCTGGGC ITCCACCCTATCGTGCCTCGTI. ATACTA

F3 N I Q Y L G F H P I V P S L L L S L L A F2 0 0 61 TTTCCTGGTCGGAAACCGTTTCGGTACATCCGTCCCGCAAGCTACCGTTTTGACTACTGA F L V G N R F G T S V P Q A T V L T T D 0 *F1 prmA BamHI 121 TAAATAAAGAGTTTTGCCATGCCTTGGATCCAACTGAAACTGAACACCACCGGCGCGAAC 1 K * M P W I Q L K L N T T G A N BglII AAGTCCTA 181 GCGGAAGATCTTAGCGATGCGCTGATGGAAGCGGGTGCCGTTTCTATCACTTTTCAGGAT 15 A E D L S D A L M E A G A V S I T F Q D AMRP TGGGTGCTATGCGGT 241 ACCCACGATACGCCAGTATTTGAACCGCTGCCGGGCGAAACGCGCCTGTGGGGCGACACC 35 T H D T P V F E P L P G E T R L W G D T 301 GATGTGATTGGTCTGTTCGACGCTGAAACCGATATGAACGACGTGGTGGCGATTCTGGAA 55 D V I G L F D A E T D M N D V V A I L E VERSE 361 AACCATCCGTCGCTCGGCGCAGGCTTCGCGCATAAA CGAACAACTAGAAGATAAAGAC 75 N H P S L G A G F A H K I E Q L E D K D 421 ;AGCGCGAATGGATGGATAATTTCCACCCGATGCGCTTTGGTGAACGACTGTGGATC 95 W E R E W M D N F H P M R F G E R L W I ART ACCGCACTACACGGCCTGCTTTTGl 481 TGCCCTAGCTGGCGTGATGT[CCGGACGAAACGCCGTCAACGTGATGTTAGATCCAGGG 115 C P S W R D V P D E N A V N V M L D P G KpnI 541 CTGGCGTTTGGTACGGGTACCCATCCAACCACCTCTCTGTGCCTGCAATGGCTCGACAGC 135 L A F G T G T H P T T S L C L Q W L D S 601 CTCGATTTAACCCGGTAAAACAGTCATCGACTTTGGCTGTGGTTCCGGCATTCTGGCGATC 155 L D L T G K T V I D F G C G S G I L A I EcoRV 661 GCGGCGCTGAAACTGGGTGCAGCAAAAGCCATTGGTATTGATATCGATCCGCAGGCGATT 175 A A L K L G A A K A I G I D I D P Q A I 721 CAGGCCCAGCCGCGATAACGCCGAACGTAATGGCGTTTCTGACCGTCTGGAACTCTACTTA 195 Q A S R D N A E R N G V S D R L E L Y L 781 CCGAAAGATCAGCCAGAAGAAATGAAAGCCGACGTGGTGGTCGCTAACATCCTTGCAGGC 215 P K D Q P E E M K A D V V V A N I L A G RUE 841 CCATTACGTGAACTGGCACCGT ATCAGCGTCCTGCCGGTTTCAGGCGGTTTGCTGGGC 235 P L R E L A P L I S V L P V S G G L L G HindIII 901 CTTTCCGGTATTCTGGCAAGCCAGGCAGAGAGCGTTTGTGAAGCTTATGCCGATAGCTTC 255 L S G I L A S Q A E S V C E A Y A D S F AL LACCTTTTTCTTCTCACCACGGCAT 961 GCACTGGACCCGGTCGTGGAAAAAGAAGAGTGGTGCCGTATTACCGGTCGTAAGAATTAA 275 A L D P V V E K E E W C R I T G R K N * 1021 CCTTCGCATCGCCGTAGGGTGACGCGGGGGCAAGTGCGAGCAAGCTCACAAAAGGCACGT

1081 AAATTTGCCGATTATTTACG;AAATTz5CGTGCCAAAATTTTCATTCATAAAGAAAAATT 1141 GAGAACTTACTCAAATTTCTTTGAGTGTAAATTTTAGTCACTATTTTCTAATATGATGAT 1201 TTTTATGAGTAATTATCGCACCACGCTCAATTTAAATGCAATTCTTTGATCCATCTCAGA 1261 GGATTGGTCAAAGTTTGGCCTTTCATCTCGTGCAAAAAATGCGTAATATACGCCGCCTTG orfI' 1321 CAGTCACAGTATGGTCATTTCTTAACTCATGCGCATCGGACAATATCAGCTCAGAAATCG 1 M R I G Q Y Q L R L R NcoI IFRO CGGTACCGACCGTAATGTCTGTCTj 1381 CCTGATCGCAGCGCCCATGGCTGGCATTACAGACAGACCTTTT 12 L I A A P M A G I T D R P F

FIG. 2. Nucleotide and deduced amino acid sequences of the prmA gene. Theboxed sequences show theoligonucleotidesused asprimers fordeterminationof the nucleotide sequence: CHIEN, VERSE, and RUEon one strand and AMRP,ART, and AL on the other. The sequence presented includes the3' end of panF(28),prmA, andthe

intergenicprmA-orflregion(8,42). Relevantrestriction endonuclease

sites are indicated. The putative ribosome-binding site ofprmA is

shaded. StarsindicatetheterminatiQncodons of panF andprmA. The black circles indicate the 5' mRNA ends mapped immediately

up-streamofprmA byS1nuclease and reversetranscriptasemapping,and

theopencircles indicate the5' ends detectedbyreversetrancriptase.

molecular mass of

31,830

Da. This is in agreementwith the

methyltransferase

of

30,000

to

40,000 Da,

obtained

by

sucrose

gradient centrifugation (4)

and

Sephadex gel

chromatography

(13). Analysis

ofthe

prmA coding

sequence shows no

signifi-cant

homology

with other

proteins

in the

DNA-protein

data bases.

However,

a

nonapeptide

motifcharacteristic of

methyl-transferases,

DXGXGXGXL

(where

X is any amino

acid),

as

described

by

Ingrosso

et al.

(26),

is found in

prmnA

between amino acids 164 and 172. Thispatternof amino acids has been observed ina

large

number of

methyltransferases specific

fora

wide range of

substrates-proteins,

nucleic

acids,

and small molecules. The

nonapeptide

motifwas found associated with two other

weakly

conserved

motifs, primarily

in the

methyl-transferases

specific

for

simple

biochemical

substrates,

e.g., amino acids

(26).

The othertwomotifsarenotpresentin the PrmA

protein

sequence. The presence of the

nonapeptide

motif is consistent with the idea thatprmA is the structural gene for the

methyltransferase

rather thana

regulatory

protein

necessary for its

activity.

The

prmA

genedoes not show the codon usage character-istic of a

strongly

expressed protein (22). Upstream

of the

ATGinitiation

codon,

there isnocomplementarity,except for

GAG,

with the 3' end of 16S RNA

(52).

However, some

homology

with the "downstream box"

(55)

associated with

high-level

expression

of certain genes is apparent, 9 of 14 nucleotidesat

positions

143to159within the

prmA

gene

(Fig.

2).

The

panF-prmA intergenic distance,

11 nucleotides, is not

sufficient foraconventionalRNA

polymerase binding

site,and

inspection

ofthis

region

showed no obvious

factor-indepen-dent

transcriptional

terminatoror

homology

with the

consen-sus -35 and -10 promotersequences,

suggesting

thatthese twogenesarecotranscribed

(see below).

The short

intergenic

distance between

panF

andprmA

might

indicate that the two

genes are

translationally

coupled.

Expression

of theprmA andpanFgenesin vivo. The lack of

obvious

transcriptional

signals

ontheDNA sequence between

panF

andprmA ledustotrytolocalize the prmA promoter

by

using

gene fusions: four

fragments

were cloned into operon

fusionvector

pRS415

to

give plasmids

pAOI,

pAO2,

pRO1,

and

pRO2 (Fig.

1). pAO2

carriesDNA from the end of accC tothe middle of

panF

(BglII site; Fig. 1)

to

give

a

panF-lacZ

fusion; pAO1

starts at the same point but extends into the

beginning

of

prmA (BamHI site; Fig. 1)

to

produce

a

prmrA-lacZ

fusion,

while

pROl

and

pRO2

carry thesame

prmA-lacZ

fusion and all

(pRO2)

or part

(pRO1)

of

panF

but lack the

sequence upstream ofthe

panF

structuralgene. The operon fusions carried

by

these

plasmids

weretransferred to

XRS45,

giving

risetoXRS/AO1, XRS/AO2, XRS/RO1, and

XRS/RO2,

which were used to

lysogenize

IBPC5321. Lysogens ofXRS/ AO1 andXRS/AO2

give

moderate 3-galactosidase

activities,

while those of XRS/RO1 and XRS/RO2 are near the

back-ground

value

(Table 3).

Thisshows thatmostprmA

expression

is

dependent

uponapromoterupstream ofpanF,which could

Thesquaresshow the 3'mRNA endsmapped downstreamofprmAby

S1 nuclease, and the major3' end is indicated bythe larger, open square.Thenonapeptidemotifcharacteristic ofmethyltransferasesis inboldface. Thesequence determinedinthis work wasfrom nucleoti-des 60to1080. Theremaining sequencewastaken fromreferences8, 28, and 42 and is included forclarity. The 3' end of the sequence determinedin this workoverlapsthatpreviously published (8, 42).Our

sequenceis inagreementwith that of Ball et al.(8), except foraG-C-G changetoG-G-C at nucleotidepositions1049 to 1051.

J.BAcrERIOL.

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TABLE 3.

,3-Galactosidase

activities instrainIBPC5321 carrying

differentpanF-lacZorprmA-lacZfusions on A lysogens

Lysogen Insert' Activity" ± SD

XRS/AO1 panFprmzA-lacZ 40 ± 2

XRS/AO2 panF-lacZ 156 ± 10

XRS/R01 'panFprmA-lacZ 1.2 ± 0.2

XRS/RO2 'panFprmA-lacZ 3.5 ± 0.3

XRS45 'lacZ 1.0 ±0.2

aAprimeindicates that the gene is truncated so that the promoter is absent.

"Activities are expressed in the units defined by Miller (39). Means of four

independentculturesareshown.

be thepanF promoter itself. The low level ofXRS/RO2 activity couldindicate that there is a weak promoter activity in the 5' region of the panF structural gene. However, it can account for only a minor amount (less than 10%) of prmA expression compared with that from the panF promoter region.

Localization of the panF promoter. The intergenic region between accC and panF is shown in Fig. 3. We used a combination of techniques, Si nuclease protection, primer extension, and in vitro transcription, to localize the panF promoter within this sequence.

The probe for SI nuclease protection experiments as the probe, PCR-generated fragment RBP22-NAPCO, which was synthesized with plasmid pAO2 DNA as the template (Fig. 4C). The NAPCO oligonucleotide was 5' end labeled with

[y-32P]ATP

andpolynucleotide kinase before polymerization.

With this probe and total E. coli RNA extracted from a

wild-type strain carrying or not carrying plasmid pFA, we

detected amajorSi-resistantDNAfragment about 340 nucle-otides long (Fig. 4A). This located the 5' end of the panF

mRNAto theseries ofTresiduesaround nucleotide 263 (Bi

of the sequence in Fig. 3). A weaker band of about 395 nucleotides which corresponds to a5' endaround nucleotide 213 (B2 in Fig. 3) was also detected. Primer extension using

B2 B1 pB32 F i'~ panF S1 mlappirgprobe RBP22 NAPCo' B2 S1resistart D tragmenis E

FIG. 4. Mapping of the 5' RNA ends upstream of panF. (A) S1 analysis.Theprobewasthe1,463-bp RBP22-NAPCOPCR-generated

fragment 32p labeledatNAPCO. Itwashybridized with RNA made

from JM109 grown in LB (lanes 2, 15 jLg; lane 3, 30 jig) or

JM109(pFA)grownin LBwith 500jigofampicillinperml(lane 4,15

jig; lane5, 30 jig)orwith30 ,ug of tRNA (lane 1).TheSI-resistant productswereelectrophoresedon a5%acrylamide-7 Mureagel.The

positions oflabeled DNA molecular size markers (inbasepairs) are

shownontheleft, and the locations of the majorprotectedbandsBI, B2, D, and E are noted. (B) Primer extension. Total RNA isolated

from either IBPC5321 (lane 1, 30 jigofRNA) orIBPC5321(pAF3)

(lane 2, 30 jigof RNA; lane 3, 15 jigofRNA) wasused for primer

extension with the 5'-end 32P-labeled NAPCO oligonucleotide. The

twomajor extension products, 340and 395nucleotides, B1andB2,are

indicated. Lanes T, G, C, and Aaresequencing reactions with pAF3

DNAand theNAPCOoligonucleotideasthe primer andrepresentthe

sequenceofthe noncoding strand. Thesequencearound themajor5' mRNAend, shown onthe right, corresponds tothesequence of the codingstrand.The5' endsofthe transcripts within thissequenceare

shownby dots. The numbers refertothenucleotidepositionsofFig. 3. (C) Diagramatic interpretationofthecombinedmapping results.

AGFIN BGFIN TGAAAACCGTGACGTGGCGATTGCCCATGTGCGCTGCAGGAGCTGCATATCGACGGTATCAAAACCAACGTTGATCTGCAGCAGATCCGCATCATGAATGACGAGAACTTCCA 10 20 30 40 50 60 70 80 90 100 110 120 ACTTTTGGCACTGCACCGCTAACGGGCGTACTTCTTACGCGACGTCCTCGACTAGTAGCTGCCATAGTTTTGGTTGCAACTAGACGTCGTCTAGGCGTAGTACTTACTGCTCTTGAAGGT E N R D V A I A R M K N A L Q E L I I D G I K T N V D L Q Q I R I M N D E N F Q accc B2 GcATGGTGGCACTAACATccAcTATCTGGAGAAAAAAcTcGGTCTTCAGGAAAAATAAGAcTGCTAAAGcGTcAAAAGGCCGGAT=ccGGCCTTTTTTATTAcTGGGGATCGACAACC 130 140 150 160 170 180 190 200 210 220 230 240 CGTACCACCGTGATTGTAGGTGATAGACCTCTT=TGAGCCAGAAGTCCTTTTTATTCTGACGATTTCGCAGT=CCGGCCTAAAAGGCCGGAAAAAATAATGACCCCTAGCTGTTGG H G G T N I H Y L E K K L G L Q E K CCCATAAGGTACAATCCCCGCTTTCTTCACCCATCAGGGACAAAAAATGGACACTCG'ITTGTTCAGGCCCATAAAGAGGCGCGCTGGGCGCTGGGGCTGACCC=TGTATCTGGCAGT 250 260 270 280 290 300 310 320 330 340 350 360 GGGTATTCCATGTTAGGGGCGAAAGAAGTGGGTAGTCCCTG=TTTACCTGTGAGCA,A*CA-AGTC-CGG-GTATTTCTCCGCGCtACCCGCGACCCCGACTGGGAAAACATAGACCGTCA FNAP TTGGTTAGTAGCCGCTTACTTATCTGGCGTTGCCCCGGTTTTACCGGCTTTCCGCGCTGGTTTGAGATGGCCTGCATCCTGACGCCGCTGCTGTTrTATTGGACTGTGCTGGGCGATGGTG 370 380 390 400 410 420 430 440 450 460 470 480 AACCAATCATCGGCGAATGAATAGACCGCAACGGGGCCAAAATGGCCGAAAGGCGCGACCAAACTCTACCGGACGTAGGACTGCGGCGACGACAAATAACCTGACACGACCCGCTACCAC AAATTTATCTATCGCGATATCCCACTGGAGGATGACGATGCAGCTTGAAGTAATTCTACOGCGGTCGCCTATCTGGTGGTGGTGTTCGGTATCTCGGTTTATGCGATGCGTAAACGGAG 490 500 T0 520 530 540 550 560 570 580 590 600 TTTAAATAGATAGCGCTATAGGGTGACCTCCTACTGCTACGTCGAACTTCATTAAGATGTrGACCAGCGGATAGACCACCACCACAACCCATAGAGCCAP TACGCTACGCACTTTGCcrc M Q L E V I L P L V A Y L V V V F G I S V Y A M R K R S panF--. NAPCO CACC 000 T

FIG. 3. ThepromoterregionofpanF. The sequence shows the 3' end of accC and thebeginningofpanFtaken from references 28 and 34. The sequences of theoligonucleotidesused in thepromoter-mappingexperimentsareboxed. Thelocations of the mRNA 5' ends(B1andB2)upstream

of panFare shownbytheblack dots. Thepossible -10 boxof promoterB1 isunderlined. A stem-loopstructure after accC is indicated by convergentarrows.Apotential ribosome-bindingsite forpanFis shaded.

1636 -1018 -517 506 396 344 298 on December 7, 2016 by INIST-CNRS http://jb.asm.org/ Downloaded from

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7184 VANET ET AL.

reversetranscriptase and the5'-end-labeled NAPCO

oligonu-cleotideontotal E.coli RNAextractsdetected 5' mRNA ends at the same positior. as found by SI protection experiments

(Fig. 4B, lanes I to3).

The 1,463-bp PCR-synthesizedprobe used in these

experi-mentscarried plasmid-derived DNAsequences at the RBP22

oligonucleotide end, which are not homologous to mRNA transcripts in this region. Protection of fragment D, with a

length of 1,028 nucleotides and corresponding to the accC panF region of the labeled probe, demonstrated thatthere is

somereadthrough from the upstream (accBC) operon.

Quan-titation ofthese bands in mRNA from a plasmid-free strain

suggests that readthrough from accBCcan account for 10to

20% of panFtranscription.

Inspection of the sequence of Fig. 3 shows that the 5' end

localized to nucleotide 263 (position Bi) is preceded by a

possible -10consenstssequence,TACAAT(nucleotides 250

to 255), but no sequei 2e homologous to the -35 consensus

sequence is located at a reasonable distance (16 to 19 bp)

upstream. The second 5' mRNA end corresponding to the

longer transcriptwaslocatedatnucleotide 213 (position B2).It fallsnear a series of T's at the base ofastem-loop structure which could be a transcription terminator for the upstream accC gene. Reverse transcriptase, notoriously sensitive to

secondarystructureswithin an mRNA, might stop atthis site and produce an artifactual 5' end. The fact that the same 5'

endwas detectedby theS1 protectiontechnique,which is less

sensitive to secondary-structure artifacts, suggests that the 395-nucleotide band detected does, indeed, correspond to a

discrete mRNA species present in vivo. Since we found readthrough transcripts from accBC into panF, it is possible that the B2 RNA is derived from these longer transcripts rather thandue to adenovo transcription start.

Toresolve thisproblem,weperformed in vitro transcription

with [ot-32P]UTP onthe unlabeled AGFIN-NAPCO fragment

(Fig. 3). Thetranscription products observedareshown inFig.

5A, lane 1: the strongest transcript (band A) is about 220 nucleotides long, theone nearthetopof the gel (bandC) is a

copy of the full-length template, and two or three RNA

transcripts of 350 to 420 nucleotides (position B) are also

evident.

The 220-nucleotide RNA (A) is a transcript made in the

opposite direction (compared with the direction of transcrip-tion ofaccC,panF, andprmA). Thiswasdemonstrated by using

another PCR-generated fragment between oligonucleotides

BGFIN and NAPCO asthetemplate for in vitro transcription

(Fig. 3). Band Awasreplaced byaband 34 nucleotides shorter.

Theclusterof RNA transcripts ofabout350to420nucleotides

(B) was unaffected, while the full-length template transcript

(C) became slightly shorter (data not shown). These

experi-ments localized the 5' end of the backward transcript to

aroundnucleotide 220onthesequenceof Fig. 3,i.e., within the

putative transcription terminator for accC. We are currently investigating whether this transcript exists in vivo.

Transcripts in the size range of350 to 420nucleotides (B)

areconsistent with the size of theS1-protected fragments and

primer extension products (340 and 395 nucleotides) detected with in vivo mRNA. To confirm that the in vitro transcripts found in position BonFig.5A, lane 1,really correspondtothe 5' ends mapped in Fig. 4,we performed reverse transcriptase

mapping ontheunlabeled products of in vitro transcription of

the AGFIN-NAPCO template. The primer used was the 32P-labeledFNAPoligonucleotide, which hybridizesnearer to the panF promoter region than does NAPCO (Fig. 3). The primer extension productsobtained areshown on Fig. 5, lane

2. Two major bands about 64 and 112nucleotides long, which

C -> A -> 1 p:: 147 = 622 527 404 309 242 238 -217 201 190 180 160 147 123 = 110 90= 76 = 67-_ 123 110 34=I =90 2 a sC 4:i IEit I B2 <- Bl accC (?F F panF ---4-- DNAtemplate

AGFIN FNAP NAPCO

A _2 RNAsynthesized

~~~~~~~~&C invitro

FIG. 5. Determination of the promoter upstream ofpanFby in

vitro transcription. (A) Lane 1, in vitro transcription on the

PCR-generated NAPCO-AGFIN fragment with[a-32P]UTP. The originof thethreemajor transcripts (A, B, and C)isdiscussed in thetext.Lane

2,unlabeledinvitrotranscriptionproductsfromtheNAPCO-AGFIN fragmentwere subjected toprimer extension performed with5'-end

32P-labeled oligonucleotideFNAPasthe primer. The extension prod-ucts(BI andB2)areindicated. Themolecular sizes (in basepairs)of labeled DNAmarkers(pBR322 digested with MspI)areindicated.(B) Diagramatic representation ofthe transcription results.

correspond to start sites around nucleotides 261 and 212 on Fig. 3,werefound. Thesetermini agreeextremelywell withthe location of the 5' ends,Bi and B2, detected previouslyon in vivo-synthesized mRNA. These data are consistent with the existence oftwopromoterregions for theexpression ofpanF. Thestronger startsite in vivo and invitro, BI,coincideswith a sequence showing homologyto the -10 consensus sequence

and seemslikelytocorrespond to arealpromoter.

Our datado notallowus tobe categorical in ourexplanation

of the B2 transcript. The lack of any promoter consensus sequLence and its location within a stem-loop structure makes

us suspect that B2 is derived from the accBC readthrough transcripts, either as a result ofendonucleolytic processingor as an experimental artifact due to thesecondary structure in the region. Such a structure could well cause reverse

tran-scriptasetoabort prematurely. This isequallypossible onthe

in vivo- and the invitro-synthesized transcriptsusedhere.The

in vitro transcription experiment produced an appreciable

amount of a full length, end-to-end transcript (C in Fig. 5A,

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12 3 4

-i

IJ

I

536 H 4-490 G 4-460 F3*4 F3205 +4- 405F2 380 F1 i

ThT-]]

_D)UG18

Fp_anF

IprmApm

. - - Simappingprobe

REV17 ART'

4-MP reversetranscriptase primer AMRP*

F2 S1resistant

F3 fragments H I

FIG. 6. Mapping of the 5' mRNA ends upstream of prmA. (A)SI analysis.Theprobe used for S1mapping was the 536-bp REVI 7-ART PCR-generated fragment 32p labeled at the ART 5' end. It was hybridized with RNA extracted from strain JM109 (lane 2, 30 ,ug) or

JMI09(pAF3)(lane 3, 15jig;lane4, 30jig)orwith tRNA (lane 1, 30

jig).

TheSI-resistantfragments were electrophoresed on a 6%

poly-acrylamide-7Mureagel. Thepositionsoflabeled DNA molecular size markers(in base pairs) are shown on the left. The lengths of protected DNAfragments Fl,F2, F3, G, and H are indicated on the right. (B) Primerextension. RNA (15 jiginlane 1 and 30 jLgin lane2) isolated from JM109(pAF3)wasused for primer extension analysis with 5'-end 32P-labeledoligonucleotideAMRP astheprimer. Lanes A, C, G, and

T are sequencing reactions with pAF3 DNA and oligonucleotide AMRP as the primer; the sequence corresponds to the noncoding strand. Molecular size markers (pBR322 digested with MspI) are

shownontheright in base pairs, and the sizes of the major extension products areindicatedontheleft. (C) Diagramaticrepresentation of the combined results.

lane 1) in which the stem-loop structurecould also form and block reverse transcriptase. Whatever itsorigin, B2isaminor componentcomparedwith the Bi transcript.

The prmA transcript. The data from the fusions panF

prmA-lacZ,panF-lacZ, andprmA-lacZ suggest that panF and prmA are cotranscribed. We looked for the polycistronic

mRNAbySI mapping withalongprobe covering bothgenes. We actually detectedverylittlebicistronic panF-prmA mRNA

but did find evidencefor mRNA5' endsimmediatelyupstream

of prmA.Toconfirm theexistenceoftranscriptstraversing the panF-prmA junction and localize the 5' mRNA ends

immedi-atelyupstreamofprmA,anS1mappingstrategysimilartothat

employed to identify the panF mRNAs was used. SI probe

REV17-ART, labeled at the ART oligonucleotide, carries

plasmid-derived sequences at the REV17 end which do not hybridize to transcripts from the panFprmA region of the chromosome(Fig.6C).Useof thisprobeandmRNAextracted

from JM109carryingor not carryingplasmid pAF3 produced

aseries ofprotectedfragmentswithlengthsof about 380

(Fl),

405(F2), 460(F3), and 490(G) nucleotides, aswellas aband

corresponding to the reannealing of the

probe (H).

Some

additional, shorterbandswerealsoobservedwith mRNA from

apAF3-carrying strain (Fig. 6A).The G bandcorresponds to

protection of the entirepanFprmA region of the probe and is

therefore equivalent tothebicistronicpanF-prmA transcript.

Reverse transcriptase mapping with the 5'-end-labeled

AMRPoligonucleotideas theprimer (Fig. 6C)produced four transcripts in the sizerangeof 125to150nucleotides (approx-imate sizes: 125, 130, 140, and 145 nucleotides) plus a longer transcript of205nucleotides(Fig.6B, lanes 1and2).These5' endswere precisely located on the sequenceofFig. 2.

Com-parison with the results of theSI experiment(Fig. 6A) showed that

SI-protected

fragment Fl corresponds to the 125-nucle-otide reverse transcriptase product, the 145-nucleotide band

correspondstoF2, and the 205-nucleotide bandcorrespondsto F3. The 130- and 140-nucleotide transcripts were always detected by primer extension, but their relative intensities varied between experiments, suggesting that they could be artifacts duetosecondarystructure.Todetermine whetherany

of these 5' ends correspond to promoters, we performed in

vitrotranscriptiononpurified DNAfragment CHIEN-AMRP.

We were not able to detect any discrete band, except one

corresponding to the full length of the template (data not shown). Since no significantpromoteractivitywasdetected in vitro or in vivoin this region,we conclude thatthe 5' ends of

mRNAs

Fl,

F2, and F3are generated byinvivoprocessingof

thepanFprmA transcript.

Comparison of the intensities of the three

SI-protected

fragments,

Fl,

F2, and F3, with that of the Gband,

represent-ingthebicistronicpanFprmAmRNA,suggeststhat75% of the

chromosome-derived transcripts are processed (Fig. 6A, lane 2). In pAF3-derived transcripts (Fig. 6, lanes 3 and 4) the

processed-to-bicistronic

transcript ratio decreased to 50%,

possibly because of limiting amounts of the processing

en-zymes.

3' mRNA extremities downstream of prmA. The DNA sequence downstream of

prmA

(Fig. 2) isvery AT rich

(72%

AT if considered from the stop codon ofprmA to the first codon of orfl, of which the central 220 bases are 78% AT) compared with theprmA structural gene

(54%

GC).There is no strong hairpinstructure followed by a series ofTresidues characteristic of prokaryotic terminators in the intergenic

prmA-orfl

region.Twopromoter sequences,atleastsixstrong

factor for inversion stimulation (Fis)-binding sites, and a cAMP receptor protein

(CRP)-binding

site have been

local-ized in this AT-rich region, and the DNA exhibits inherent

curvature (8, 42).

We used

SI

mapping to determine the fate ofprmA

tran-scripts after the structural gene. The fragment used covered the 3' end ofprmA andwaslabeledatthe

IHindIII

site

(Fig. 2).

The other, unlabeled end was located within the

orfi

gene

(corresponding

to

oligonucleotide IFRO; Fig. 2).

When

mRNA froma

plasmid-bearing

strain

[IBPC5321(pAF3)]

was

used to enhance the amount ofprmA

transcripts,

this

probe

detectedamajorbandofabout 185nucleotides andaseries of longertranscripts (Fig. 7A, lanes4 and 5). The same 3' ends weredetectablein RNAextracts

prepared

froma

plasmid-free

strain

(Fig.

7A,lane 6).The major3' end

(open

squarein

Fig.

2)is located100nucleotides downstream of theprmAgeneand

mapstothe distal side ofa small

palindromic

sequencewhich isnot,however, followedbyaseries ofTresidues

(nucleotides

1098 to 1111 of the sequence in

Fig.

2).

The ladder of bands

corresponding tolonger

transcripts

of200to 250 nucleotides showsa

periodicity

ofabout 10nucleotides.

Thus,

these 3'ends onthe mRNA

correspond

to

positions

onthe DNA

template

separated byaboutone turnofthe B DNAhelix

(10.5

bp).

This

spacing

suggests that 3' endsare

generated

by

periodic

termi-nation of

transcription

at

in-phase

positions

ontheDNA.Since

this

region

is

inherently

bent because of the presence of

http://jb.asm.org/

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7186 VANET ET AL.

1 2 3 4 5

HindlII

E . ',~~~~~~~~~~~~~~~~~~~~~

6 M transcribe through the intergenic prmA-orfl region into the

orfl-fis

operon.

However,

the contribution of

panF-prmA

transcription to orfl-fis expression in vivo is not likely to be verysignificant invivo.

517 506 396 344 298 220 201 154 134

|

panF IprmA

|

-

OS

|

V CRP V IVIII Hindlill 4. Simappin( I IFRO probe S1 resistant fragments i-I -I II

FIG. 7. SI analysis ofthe 3' RNA endsofprmA. (A) The probe usedwasthe477-bp HindIll-IFRO fragment3' 32Pendlabeledatthe

HindIll end. Theprobewashybridizedwith 15or30 jLgof RNAfrom

IBPC5321(pBR322) (lanes 2 and 3), 15 or 30 jLg of RNA from

IBPC5321(pAF3) (lanes4and5),50jLgof RNA fromIBPC5321(lane

6),or30 jLgof tRNA(lane 1).TheSI-resistantproductswereanalyzed

on a5%polyacrylamide-7Mureagel.Thepositions ofmolecular size

markers are indicatedon the right inbase pairs,and the sizeof the

major SI-resistant fragment is given on the left. (B) Diagrammatic

representation of theexperiment.CRP,cAMPreceptorprotein

bind-ing site; ItoVI,Fisbinding sites.

poly(A) tracts and has several Fis-binding sites and a cAMP

receptor protein-binding site (Fig. 7B), which are known to

affectDNAstructure(8),wesuspectthat the 3' endsobserved hereare related tothe topology of the region rather than due to classical transcription termination. Analysis of the mRNA

fromthepAF3plasmid-carryingstrain revealed protection ofa

fragment just shorter than thefull-length probe (Fig. 7, lanes 4 and 5). This isbecause the insert in pAF3, which endsat the NcoI site, is slightly shorter than the SI probe which was

synthesized with oligonucleotide IFRO (Fig. 2). The

protec-tion of thisshorterfragment shows thatacertainpercentageof RNApolymerase moleculesontheplasmid DNA templatecan

DISCUSSION

Althoughcotranscription isaconvenient strategy for coregu-lation of genes with a common function, there are acertain number ofexamplesofcotranscription of genes withunrelated

functions,

e.g., the rpsU dnaG rpoD operon that encodes

ribosomal protein S21, DNA primase, and RNA polymerase

subunit sigma 70 (12). In thiswork, we show that the gene necessary for posttranslational modification of a ribosomal

protein-the methyltransferase ofLi1-is cotranscribed with the gene for pantothenate permease. Pantothenate is a

pre-cursorfor coenzyme A. The genepanFisnecessaryonlywhen

pantothenatemustbetaken up from theenvironment,because

wild-type E. coli can synthesize pantothenate from 1-alanine and pantoateby usingthe enzyme encodedby panC (see Fig.

I inreference 62). Weare currently investigatingthe

implica-tions of coexpression of these two genes, panF and prmA,

which have verydifferent functions.

prmA is the first gene that controls the methylation of a

ribosomal protein to be sequenced. A mutation (prmB) that affects themethylationofL3 has beenisolated and mappedto

50minonthe E.coli chromosome (14, 37).We are unaware of

information, either genetic or biochemical, concerning the other ribosomal protein methyltransferases. The genedosage

effect on L1I methyltransferase activity observed with the

prmA gene carried on amulticopy plasmid, the fact that the size ofthe protein encoded by prmA agrees with themolecular weight of the partially purified enzyme (4, 13), and the presence ofashort amino acid motif found in many methyl-transferases ofdifferent specificities (26) argue thatprmA is the structural gene for LII methyltransferase. A detailed

analysisofmutations within the prmA gene and their effecton

LII methylation also support this conclusion (64).

The prmA ORF appears to start at the ATG codon I1

nucleotides downstream of thepanFtermination codon. Pro-teinfusionswith a truncated lacZ gene were constructed at the

BamHI site 11 nucleotides downstream of this ATG. The

hybrid gene which carries the first four codons of prmA in phase with the lacZ gene, when present on a plasmid or bacteriophage A vector, produces blue colonies on 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside (X-Gal) plates,

show-ingthat the first ATG doesfunction as an initiation codon(63).

A second ATGcodon, 22 amino acidsdownstream, shows no

betterribosome-bindingsite. The short distance betweenprmA andpanF, together with the lack of any recognizable Shine-Dalgarno sequence, exceptGAG, suggests thatthe twogenes aretranslationallycoupled. Efficienttranslationalcouplinghas been observed in various bacterial systems in which the termination and initiation codons canoverlapor beseparated bytensof nucleotides (1, 24,36).

The datapresentedhereshowthat, bothinvivoandinvitro, genes panF and prmA are cotranscribed from a promoter locatedupstream of the panFstructural gene.Thebest candi-date for the panF promoter isthatwhich gives risetotranscript BI in Fig. 3. Transcripts from BI carry a 255-nucleotides

untranslatedleaderupstreamof the panFstructuralgene. This long mRNA leadermight be involvedin regulationof expres-sion of theoperon,althoughwehavenotidentifiedany obvious

regulatory elements in thisregion.

We alsodetected, by different techniques, a5' mRNA end located at the base of astem-loop structure (B2inFig. 3). Our 185 J. BAC-FERIOL. lr ,., ig on December 7, 2016 by INIST-CNRS http://jb.asm.org/ Downloaded from

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present data cannot unambiguously assign this 5' end to a promoteractivity. If it doesnotcorrespondto apromoter,then it derives from posttranscriptional processing of the longer transcript whichwas detected in small amounts. These longer transcripts presumably initiate from the upstream accBC operonandmust readthrough theputative terminator located afteraccC (nucleotides200 to 220 inFig. 3), which in thiscase

could serve as an mRNA-processingsite.

Another potential regulatory device is suggested by the detection of a promoter activity in vitro thatgives rise tothe synthesis ofadivergentmRNAinthissameregion. The length of thetranscript detected (AinFig.5A, lane 1)locates itsstart

pointvery near to B2(Fig. 3) butontheopposite strand.There are no sequences strongly homologous to the consensus -35 and -10 promoter sequences in thisregion.We are currently investigating whether this promoter is functional in vivo and what the length of the putative antisense mRNA is. It is interesting that there is a long ORF of 314 amino acids downstream ofthis promoter,completely contained within the accCstructural gene but expressed from theopposite strand.

Despitethelack ofanypromoteractivity, in vivoorinvitro,

expressing a monocistronic prmA transcript, we did locate several mRNA 5' ends upstream of theprmA structural gene

which lie mostly within the 3' end of the panF structuralgene

transcript. The existence of a discrete prmA transcript was suggested by Northern (RNA) analysis (63). The use of a probe internaltotheprmA genedetectedbasically asmearof hybridizing material with lengths ofup to 1,800 nucleotides. There was also a rather diffuse band of about 1,200 nucleo-tides. This is sufficient to cover the entireprmA gene but not bothpanF andprmA. The use ofapanF-specificprobe failed

to detect this or any other discrete bands. The simplest

interpretation of these results is that thebicistronicpanF-prmA

transcript is subject to posttranscriptional processing which generates an mRNAwhichjustcoverstheprmA gene. Weare currentlyinvestigatingthishypothesis. Itisinterestingthatany

endonucleolytic processing within the 3' end of the panF

mRNA will generate a truncated panF mRNA incapable of directing the synthesis of a full-size PanF protein. The

n-ga-lactosidase activities of the operon fusions (Table

2) provide

some indication of the relative expression levels of the two genes. The lower level of ,B-galactosidase

activity

of the

prmA-lacZoperonfusion than of thepanF-lacZoperonfusion

suggeststhatsomedown regulationof

expression

occurs.This effect could be relatedtotheputative processingeventsthatwe

have detected in thepanF-prmA mRNA.

ACKNOWLEDGMENTS

We thank Marie-France Guerin for performing the methylation tests,RichardBuckinghamandMathiasSpringerforcriticalreadingof themanuscript, MarianneGrunberg-Managoforconstantinterest,P.

LeChien for encouragement,SylvieBlanda forpatience,andareferee forconstructivecriticism.

This workwassupported bygrantsfrom theCNRS, INSERM,Paris 7 University, the A.R.C., and the Fondation pour la Recherche

Medicale.A.V. wassupported byagrantfrom theMRT.

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