Nucleotide sequence of the glnA-glnL intercistronic region of Escherichia coli

91

Gene, 37 (1985) 91-99 Elsevier GENE 1346

Nucleotide sequence of the glnA-glnL intercistronic region of Escherichiu coli (Recombin~t

DNA; gtnALG operon; glutamine synthetase adenylylation site; te~ination;

REP sequences)

Mario Rocha, Martha Wzquez, Alejandro Garciarrubio aud Aiejandra A. Covarrubias* Gentro de Investigacibn sobre Fijacitin de Nitrcigeno, Universidad National Autcinoma de MPxico, Apartado Postal 565-A, Cuernavaca, Morelos (Mexico) Tel. (9073) 139-877 (Received January 4th, 1985) (Revision received February 20th, 1985) {Accepted March lst, 1985)

SUMMARY

The nucleotide (nt) sequence of a 6X2-bp fragment containing the 3’ end of the glnA gene, the region between the glnA and glnL genes, and the 5’ end of the glnL gene from Escherichiu coli was determined. This segment contains the region coding for the last 107 amino acids (aa) of glutamine synthetase, including the adenylylation site of this enzyme. The analysis of this sequence revealed two REP sequences, a Rho-independent terminator, the putative ghzL promoter and the possible binding site for the gtnG product, NR, .

INTRODUCTION

In enteric bacteria the synthesis of GS, as well as that of many proteins that transport or degrade nitrogen compounds (Ntr systems), is regulated in response to the quality and abundance of the nitrogen source in the growth medium (Woolfolk et al., 1966; Prival and Magasanik, 1971; Prival et al., 1973; Kustu et al., 1979a; Magasanik, 1982). Tbis regulation seems to be exerted at the level of transcription through the products of the glnF, glnG * To whom correspondence addressed.

and reprint requests should be

Abbreviations: aa, amino acid(s); bp, base pairs; GS, glutamine synthetase; IPTG, isopropyl-fi-D-thiogalactoside; NR, , nitrogen regulator; nt, nucleotide(s); Ntr, nitrogen-regulated; PolIk, Klenow fragment of the E. cc& DNA polymerase I; RBS, ribosomebinding site; REP, repetitive extragenic p~indromi~; Xgal, 5-brom~-chloro-3-indolyI-~-~g~actoside. 0378-l 119/85/$03.30 0 I985 Eisevier Science Publishers

and g&L genes (for reviews see Magasanik, 1982; Merrick, 1982). Evidence from different laboratories suggests that these gene products function both positively and negatively (Garcia et al., 1977; Kustu et al., 1979a; Pahel and Tyler, 1979; McFarland et al., 1981; MacNeil et al., 1982a; Alvarez-Morales et al., 1984). Both g&G and g1n.i.together with g&A, the glutamine synthetase structural gene, form a complex operon (Pahel et al., 1982; ~ajewska-G~nkiewicz and Kustu, 1984; Alvarez-Morales et al., 1984), whereas the glnFgene is unlinked (Garcia et al., 1977; Pahel et al., 1978; Gaillardin and Magasanik, 1978; Leonardo and Goldberg, 1980). It has been shown that the transcription of the glnALG operon proceeds through glnA, glnL and glnG in that order (Rothstein et al., 1980; Backman et al, 1981; MacNeil et al., 1982b; Pahel et al,, 1982; Espin et al., 1981; Krajewska-G~nkie~~z and Kustu, 1983). The transcription of g&G can be initiated at the promoter for

glnA as well as at a site located glnA (MacNeil processes

downstream

exerted by the pro-

duct of glnG (NR,). This gene product scription et al.,

or absence

initiated

1982;

represses,

of the glnF product,

at the downstream

Backman

et al.,

1983;

et al., 1983; Reitzer and Magasanik, ber of observations region, with promoter

have

suggested

in

tran-

and ligation reactions

to Maniatis

was as in Messing

were performed

et al. (1982). Transformation

et al. (1981).

(d) Nucleotide sequencing

1983). A numthat

a DNA

activity, is localized

between

1982; Covarrubias

Restriction according

site (Pahel Ueno-Nishio

gfnA and glnG (Chen et al., 1982; Goldie and Magasanik,

(c) Cloning procedures

et al., 1982b; Pahel et al., 1982). Both

are subject to control

the presence

from

and Bastarrachea,

Nucleotide

sequencing

was carried out using the

chain termination procedure (Sanger et al., 1977; Messing et al., 198 1; Messing and Vieira, 1982).

1983).

Ueno Nishio et al. (1983) have located a DNA region with the functional properties of a promoter and operator in a 270-bp DNA fragment. This fragment lies at the end of, or distal to, glnA and contains the site at which translation of glnL is initiated. One of our approaches to understand the control of the gInALG operon has been the analysis of its regulatory regions. In this study, we report the complete nucleotide sequence of a 682-bp BarnHI-CfuI fragment containing the 3’ end ofgInA, theglti-glnL intergenic region, as well as the 5’ end of glnL from E. coli.

(e) RNA isolation The MX614 strain was grown on glucose NN minimal medium (Covarrubias et al., 1980b) with glutamine 1 mg/ml as nitrogen source. The culture was harvested at Aeoo = 0.5, and total RNA extracted according to Aiba et al. (198 1).

RESULTS

was

AND DISCUSSION

(a) Sequencing strategy

MATERIALS

AND METHODS

(a) Reagents, media and enzymes Electrophoresis reagents were from Bio-Rad, deoxynucleotides from Sigma Chemical Co., and dideoxynucleotides from PL-Biochemicals. Deoxyadenosine 5-[ a-35S Jthio-triphosphate was purchased from Amersham International. IPTG and Xgal were purchased from Sigma Chemical Co. All the other reagents and media were of analytical grade. Restriction endonucleases, T4 DNA ligase and PolIk were purchased from commercial sources. (b) Bacterial strains and cloning vehicles The E. coli K-12 strain, JMlOl (Messing et al., 1981) was used as host for vector phages M 13mpS and M13mp9 (Messing and Vieira, 1982). The wildtype strain used for RNA isolation, MX614, was described previously (Osorio et al., 1984).

As mentioned above, it has been shown that the presence of a promoter-operator region in a portion of the glnALG operon located between glnA and glnL intercistronic region should be contained in the 691bp BamHI-EcoRI fragment from plasmids pACR.5 (Covarrubias and Bastarrachea, 1983) (Fig. 1). To obtain the nt sequence of the gInL 5’-control region, this BarnHI-EcoRI fragment was cloned in phages M 13mp8 and M 13mp9. The nt sequence of the two ends of this fragment was determined. Fragments of about 300 bp from the glnALG region obtained by sonication (Deininger, 1983) were cloned into SmaI-digested M13mp8. Appropriate clones were identified by dot hybridization (Maniatis et al., 1982) using the 682-bp BamHI-C/u1 fragment (Fig. 1) as probe. At the same time, the purified BamHI-C/a1 fragment was digested with HinfI. The three resulting fragments of 283, 177 and 222 bp were filled-in using PolIk, and cloned into M13mp8 digested with SmaI. These clones allowed us to determine the sequence of both strands of the BamHI-ClaI fragment by the Sanger chain termination procedure (Sanger et al., 1977). The nt sequence obtained is shown in Fig. 2.

93

Fig. 1. Physical map of the gin genes of E. coli and of the pACR5 plasmid. (A) Restriction map of the glnALG operon. The black bar represents the BarnHI-CloI fragment which was sequenced. The positions ofglnA,glnL andglnG are shown by the open bars; the open arrows indicate the direction of transcription. Some of the restriction sites are indicated; the vertical arrows show the Hinfl sites internal to the Bum HI-ClaI fragment. The map positions of the genes are based on the M,s of the peptides synthesized by minicells containing plasmids with multiple deletions of the glnALG DNA region (not shown). The locations assigned to these genes are in agreement with those reported in the literature (Backman et al., 1981; Pahel et al., 1982; Chen et al., 1982; Ueno-Nishio et al., 1983). (B) Map of the pACR5 plasmid (Covarrubias and Bastarrachea, 1983). The dark bar represents DNA from pBR322. The single line indicates DNA derived from wild-type E. coli.

While this work was in progress, Ueno-Nishio et al. (1984) reported part of this sequence, comprising the region from position 281 to position 678 (Fig. 2).

(c) The glnA-gZnL intercistronic region Analysis of the glnA-glnl intercistronic shows four interesting sequences.

region

(b) The C terminus of glutamine synthetase From the first 322 nt we deduced the aa sequence of the C terminus of GS, as well as its adenylylation site. The deduced sequence agrees completely with the known seven C-terminal aa of GS (Ginsburg and Stadtman, 1973). Furthermore, the aa sequence around the adenylylation site, indicated in Fig. 2, is almost identical with that obtained through peptide sequence by Heinrikson and Kindgdon (1971). However, some modifications have been introduced to the reported aa sequence (R.L. Heinrikson, personal communication) which makes it identical to the deduced aa sequence given here. In Fig. 3 the deduced aa of this region obtained from E. coli and Salmonella typhimurium (Hanau et al., 1983; J. Brenchley, personal communication) were compared. Two regions are highly conserved. One, encoded from positions 92-163, has the adenylylation site; the other, encoded from positions 260-325, contains the C terminus. This could be relevant in delimiting the functional domains of the enzyme.

(1) The REP sequences Two regions are highly homologous to the consensus REP sequence (Stern et al., 1984). One of them is located in the upper strand at positions 395-430, and the other is located in the lower strand at positions 423-454 (Fig. 2). The localization of the REP sequence found here is similar to that found in various operons from E. coli and S. typhimurium (Stem et al., 1984). In Fig. 4 the REP sequences we found are compared with the consensus (Higgins et al., 1982a; Stem et al., 1984). An 8-bp overlap exists between the two REP sequences. Two possible RNA secondary structures can be formed which have a free energy of -23.9 kcal/mol and -28.5 kcal/mol, respectively (Fig. 4). This suggests that either one of these structures can be formed in vivo. Stem et al. (1984) proposed that the REP sequences are involved in chromosome structure and organization. We do not know as yet if the sequences described here play any role in regulating the expression of the glnALG operon. However, the analysis of

94

so

. . .

. . .

. . .

. . . TTG GCC ATG G:\C ACG AAA CGA CGC GAC GAC ‘TAC CCG CCA G/U 100

Lys AAG TTC

Ile XTC T:\G

His Pro CA’C CCG GT.~ Gee

Gly GGC ccG

Glu GAA CTT

.A-\ia blct GCC ATG cGG TAC

Asp GAC CTG

Lys AAA ~77

Asn AAC TTG

CT,1 CC;\

I‘AG TTC ‘I’TG

Glu GAA CTT

Lys AAA rri‘

AMP Leu CTG GAC

‘Tbr TAT ATA

Asp CAC CTG

Leu CTG GAC

Pro CCC Gee

Pro CC,\ GGT

Glu. (;,\A 07

Ala CCC CGC

? I)0

150 Pro CCA GGT

Gln cAG GTC

Glu Ile GAG ATC c,rC ‘rA(;

Val

Ala

Gly

Ser

Lcu

Glu

CAA

CGT

CCG

AGA

GAC

CTT

GTT GcA GcicII~ CTG m4

Glu GAA CTT

Ala GCA CGT

Leu 0-G GAC

Asn AAC TTG

Glu GAA CTT

I.eu CT(; GAC

Asp GAT CTA

Leu CTG GAC

Asp GAc CTG

Arg Glu CGC (;Ac GCG (:TC

Phr Leu 7’7x1 c,rG AAG GAC

l.ys AAA TTT

Ala CCC CGG

Leu Arg CTG CGT G.4C GCA

Arg CGC GCC

Glu CAA CTT

Asp GAT CTA

Arg Val CGC GTG GCG CAC

LSO Giy Giy Val GGT GGC GTG CC.4 CCG CAC Hinf

Phe TTC AAG

Thr ACT TGA

Asp GAC CTG

Glu GAA CTT

Ala GCA CGT

IIe ATT TAA

Asp CAT CTA

Ala Tyr GCG TAC CCC ATG

Ilc ATC TAG

Ala GCT CGA

Glu GAA CTT

Asp GAC CTG

350

3uo

1

Arg Met Thr Pro His CGT ATG .4CT CCG CAT GCA TAC TGA GGC GTA

Pro Val CCG GTA GGC CAT

Glu Phe GAG TTT CTC AAA

Glu GAG CTC

Leu CTG GAC

Tyr TAC ATG

Tyr TAC ATC

Scr AGC TCG

Val GTC CAG

End TAA ATT

GTGTTTTAGTTGCCGTGGAAACTTTTC CACAAAATCAACGGCACCTT’I’GAAAAG

400

4

GCCTGTCTCTGGCAGGCCTGGCATCGGTGGCAAGCACATCACGCCGGATGCGACGCA~ATGCG’rCTTATCCGGCCTA~ACG(;‘rGATG~TGT CGGACAGt\GACC(;TCCG(;f\CCCTA(;CCACCtiT’~C(;TGTAGTGCGGCC’rACGC’rGC(;T’rTACG(~A(:AA’rAG~;(:CGGA’rGTGCCACTACTACA k 450

Hinf -

I

,

500

P

1

GGTAGGCCGGr\GCAGG’;‘GAGTCGCTCTCCAACGTGA~GT’rTG’rCAG~TATCTG,r.4G~CCATC’rC’rG~A’rGG~;CTTT~TTCTCCGTC~AI.TC CCATCCGGCCTCGTCCACTCAGCGAGAGGTTCCACTTCACTTCAAACAGTCGA’rAGACA’~CGGGTAGAGA(~G’rACCCGAAAAAAGAGGCAGTTAAG

600

550

.

4

* Met Ala Thr Gl ATG GCA ACA GG TAC CGT TGT cc

CACTAAAATGGTGCAACCTGTKmACTGCTTT GTGATTTTACCACGTTGGACAAGTCCKKACGAAA

TCTGATGCTTCGCGCTTTTTATCCGTAAAAAG AGACTACGAAGCGCGAAAAATAGGCATTTTTC 650

Y Thr Gln Pro Asp Ala Gly Gln Ile Leu Asn Ser Leu Ile Asn Ser Ile Leu Leu Ile Asp c XCG CAG ccc GAT GCT GGG CAG ATC CTC AAC TCG CTG xrr AAC AGT ATT TTG TTA ATC GAT G TGC GTC GGG CTA CGA CCC GTC TAG GAG TTG AGC GAC TAA TTG TCA TAA A.. . . . . . . . . .

REP2

t,, Pg!&

Fig. 2. Nucleotide The REP sequences

sequence

of the glnA 3’ end, the ghA-glnL

are shown by the solid arrows.

by the open facing arrows.

The possible

NR, binding site. The wavy line indicates their respective Heinrikson presumptive

codons.

intercistronic

The inverted

repeat

region and the glnnt 5’ end. Numbering in the rho-independent

-10 region of the glnL promoter the putative

RBS. The deduced

is boxed. The hatched aa sequences

glnA terminator

1971). The drawing (tA), glnL promoter

below the nt sequence

shows

(PRlnL.), and NR, binding

the relative

site (O,,n,).

is from the 5’ end. sequence

bar shows location

for the correct

Symbol AMP points to the tyrosyl residue to which AMP is covalently

and Kingdon,

terminator-like

bound

location

reading

is indicated

of the postulated

frames are shown above

(Shapiro

and Stadtman,

of the REP sequences,

1968; and the

95

E.C. S.t.

CG SC

Gly

Glu

Ala

Met

Asp

Lys

Asn

Leu

AMP I Tyr

Asp

Leu

Pro

Pro

Glu

Glu

Ala

Lys

Glu

Ile

Pro

Gln

Val

Ala

GGC

GAA

GCC

ATG

GAC

AAA

AAC

CTG

TAT

GAC

CTG

CCG

CCA

GAA

GAA

GCG

AAA

GAG

ATC

CCA

CAG

GTT

GCA

tt*

l

***

***

l

**

GCG

AAA

GAG

ATC

CCA

*** CAG

* GTA

::G

Glu GAA *** GAA

Glu GAA *** GAA

Asp GAT l *t GAT

Asp GAC ttt GAC

Arq CGC *** CGC

Val GTG t** GTG

Arq CGT *** CGT

-%G

CG;

AG:

Arq

Ser

.:;:

6;:

1::

::;

:;a

;:I

k::

:;;;

:;a

::G

2::

t::

Gly

Ser

Leu

Glu

Glu

Ala

Leu

Asn

Glu

Le"

Asp

Leu

Asp

Arg

E.C.

GGC

TCT

CTG

GAA

GAA

GCA

CTG

AAC

GAA

CTG

GAT

CTG

GAC

CGC

66bp

s.t.

::T

;:;

:;z;

5:;

AG: A+g

AGC Ser

GCT Ala

TGG Trp

A:C Asn

GC: Ala

CTG Leu

AAA Lys

CT: Leu

GAA Glu

n.d.

Met

Thr

Pro

His

Pro

Val

Glu

Phe

Glu

Leu

Tyr

Tyr

Ser

Val

End

ATG

ACT

CCG

CAT

CCG

GTA

GAG

TTT

GAG

CTG

TAC

TAC

AGC

GTC

TAA

l *t

l t

l *.

l *

ttt

l *t

l **

t**

tt*

l t*

**t

t**

***

t*

l **

ATG

ACC

CCG

CAC

CCG

GTA

GAG

TTT

GAG

CTG

TAC

TAC

AGC

GTT

TAA

E.C.

s.t.

E.C.

**

172bp

Y AGCCCATCTCTGCATGGGCTTTTT

158bp

AGCCCATCCCAAGATGGGCTTTTT

**t**t**

*

CACTAAAATGGTGCAACCTGTTC

TCTCCGTCAATTCTCTGATGCTTCGCGCTTTTTATCCGTAAAAAG t**tt *** l **.** l ***t*tt*tt

l

t***t*tt***

A?%??ACTGCTTT

s.t.

E.C.

s.t.

"et

Ala

Thr

Gly

Thr

Gln

Pro

Asp

Ala

Gly

Gl"

Ile

Leu

Asn

Ser

Leu

Ile

Asn

Ser

ATG et* ATG

GCA t** GCA

ACA

GGC *a* GGC

ACG

CAG **a CAG

CCC l ** CCC

GAT l tt GAT

GCT l ** GCT

GGG l ** GGG

CAG *** CAG

ATC l ** ATC

CTC

AAC

TCG

CTG

ATT

AAC

AGT

**(t

l *

l *t

CTC

AAT

TCG

T;A

:;C

;\A;

::C

l

AGC Ser

Fig. 3. Comparison

between

l

ATA Il.2

of the glnA 3’ end, the glnA-glnL intercistronic

the nt sequences

and that of S. typhimurium (S.t.)DNAs. Only the sequences

of the antisense

strands

region and the glnL 5’ end of E. coli (E.G.)

are shown for both cases. The deduced

codons for the E. coli sequence

for the correct reading frames are shown immediately

above their respective

The asterisks

A 66-bp DNA region has not been sequenced

region

indicate

identities

in the E. coli sequence

between

sequences.

downstream

the grnA gene shows

very few homologies

S. typhimurium. For other symbols see Fig. 2. The sources ofthe S. typhimurium nt sequences for the first 113-bp, and Hanau

CONSENSU3

Gee+

ATG

aa sequences

and below for S. typhimurium.

(nd.) for S. typhimurium. A 172-bp

to the corresponding were J. Brenchley

158-bp

(personal

sequence

in

communication)

et al. (1983) for the rest of the sequence.

. cG~cG$

($GFCTTATC~GGCCTAC AG =-

Kcol mold

23.9

GAU REP2

3’-

GCCGG I 423

ATG

TGCCACTACTACACC

ATCCGGCCT-5’

t

I

:

G

C-G’ A-U C-G’

454

ig

E”c G-C 5:

C-G C-G

UAU-AGC-3’

dG = - 28-S

Fig. 4. The REP sequence with the REP consensus

in thegM-glnL sequence

of Fig. 2. Possible RNA secondary and Borer et al. (1974).

intercistronic

(Higgins structures

region. The REP sequences

Kcal mol -’

found in this region (REP1 and REPZ) are compared

et al., 1982a; Stern et al., 1984). Numbers

below indicate

are also shown. The dG values were calculated

according

their position to Cantor

in the nt sequence

and Schimmel(l980)

the nt sequence intercistronic

of the S. typhimurium

dent terminator

gInA-glnL

region

stem and loop structure

does not show any sequence to the REP sequences. This could sug-

homologous

gest that they are not relevant

for the expression

1978) with a theoretical

of

it is followed

the operon. This type of sequence is also absent from

involvement

the upstream

duction

region of the gbzA gene.

sequence

located

(Adhya

and Gottesman,

LIG of - 13.5 kcal/mol

by a row of six T’s of a termination

of transcription

process

observed

and

(Fig. 2). The in the re-

between

the g/nA

and glnG genes have been suggested (Pahel et al., 1982; Reitzer and Magasanik, 1983). The experi-

(2) A possible glnA terminator The

since it might form a G + C-rich

at

positions

(Fig. 2) has all the characteristics

498-522

ment shown in Fig. 5 demonstrates

of a rho-indepen-

gen limitation,

that, under nitro-

the main glnA transcript

z

h,

I!

is approx.

E 8 t c

0

I

1624 Fig. 5. Northern by Maniatis

blot hybridization.

hybridization

were carried

out as described

glnA, labeled with [32P]dCTP dpm/pg.

of agarose

by Thomas

by nick translation.

gels containing

was added

2.2 M formaldehyde

to all buffers.

rotavirus

(SAE

The probe was used at a concentration

The transcription

M. Rocha,

P. Leon, F. Bastarrachea structure

et al., 1980a; Osorio wavy lines indicate

Arrows indicate

markers:

and A.A. Covarrubias,

submitted

here. The restriction

et al., 1984). The initiation the DNA and the mRNA,

for publication).

sites were localized

Distances

through

filter

internal

to

of 5 x 10’

et al., 1984) was hybridized

in the lower part shows primer extension

RNA from

the predicted

glnA

(A. Garciarrubio,

The stop codon (TAA) and the Rho-independent by conventional

codon (ATG) was taken from Covarrubias respectively.

MX614 (Osorio

2904 nt and 1541 nt, and denaturated

start point (symbol I at the left end of the map) was obtained

(r+,) are described

fragment

of 10 ng/ml and a specific activity

strain

E. colirRNA:

were as described

as well as the nitrocellulose

the 700-bp BarnHI-EcoRI

11): 3700 nt, 1620 nt, 1350 nt, 1120 nt and 660 nt. The scheme

transcript.

terminator-like

fragment.

and electrophoresis

The gel transfers

(1980). We used as a probe

4 pg of total RNA were used per gel slot. Total RNA from the wild-type

against the EcoRI-BumHI-generated human

Preparation

et al. (19X2), except that 2 mM NaKHPO,

restriction

and Bastarrachea

are in bp and the size of the transcript

mapping

(Covarrubias

(1983). The straight is in nt.

and

97

1640 nt long. The existence of a largely preferred site for transcription termination is also suggested since transcripts of other sizes are not evident. Assuming an average M, of 120 per aa residue, the coding requirement for the GS monomer (M, = 55 000; Bender and Streicher, 1979) is approx. 1375 bp. With these considerations in mind, the 3’ end of the 1640-nt transcript should be located not farther than 300 bp after the GS stop codon. A more precise calculation based on the exact location of the 1640-nt transcript 5’ end (A. Garciarrubio, M. Rocha, P. Leon, F. Bastarrachea and A.A. Covarrubias, submitted for publication) allowed us to localize the 3 ’ end of this transcript within 40 nt of the site where the terminator-like sequence is found. Although a more detailed study is needed it seems probable that this sequence corresponds to a functional terminator. This terminator sequence shows high homology with the one found in the S. typhimurium intercistronic region (Hanau et al., 1983; Fig. 3). However, the structure found in S. typhimurium should be more stable than the one found in E. coli since the theoretical free energy of the former is -16.1 kcal/mol. (3) The presumptive glnL promoter Genetic data strongly suggest the existence of a promoter between glnA and glnL (Chen et al., 1982, Goldie and Magasanik, 1982; Ueno-Nishio et al., 1983). Indeed we found a conserved Pribnow box and a putative -35 sequence in this region (from positions 542-573). Recently, Ueno-Nishio et al. (1984) have reported the location of the glnL transcription start site at position 579,6 bp downstream from the conserved Pribnow box shown in Fig. 2. This supports the role of this sequence (TATAAT) as part of the glnL promoter. -..+

c-c-

l

5’. TTGCACCAACATGGTGCTTAATGmGAAGCACTATATTGGTGCAAC ..**** . t.fft.ff. ..**, .f ..*..*, 5’. TGCACTAAAATSGTGCAAC 5’. TGCACTAAA>ITGGTGCAAC -3’ **577, 191 511 -.

Fig. 6. Comparison

between

present

and glnL control

sequence

in the &A

above corresponds

bias and Bastarrachea,

the possible

below refer to the positions

binding

sites

The nucleotide

to theglnA control region (Covarru-

1983). The asterisks

with the 19-mer found in the glnL control dyad symmetries.

Plnl

591

NR,

regions.

UN&

3’ -3’

indicate

identities

region. The numbers

in Fig. 2. The arrows

indicate

the

The -10 region of one of the gInA promoters

is boxed (A. Garciarrubio, and A.A. Covarrubias,

M. Rocha, submitted

P. Leon, F. Bastarrachea

for publication).

(4) A possible NR, binding site A 19-bp sequence that contains an inverted repeat was found close to the presumptive glnL promoter (from positions 573-591). The same sequence is also found in the glnA control region as shown in Fig. 6. In addition, this sequence is also present in the S. typhimurium dhuA promoter (Higgins et al., 1982b), which is subject to nitrogen regulation (Kustu et al., 1979b; Higgins and Ames, 1981; Stern et al., 1984). The fact that the glnL as well as the glnA promoters are repressed by NR, (Goldie and Magasanik, 1982; Ueno-Nishio et al., 1983; Wei and Kustu, 1981; Pahel et al., 1982) is supported by data which show that NR, is able to bind specifically to a DNA fragment containing the glnL promoter (Reitzer and Magasanik, 1983) or the glnA promoter (A. Garciarrubio, M. Rocha, P. Leon, F. Bastarrachea and A.A. Covarrubias, submitted for publication). In addition, the finding that a DNA fragment containing this 19-bp sequence is protected from DNase I digestion (Ueno-Nishio et al., 1984) further supports the role of this sequence in DNA recognition by NR, . Interestingly, we found two possible NR, binding sites in the glnA control region. These two 19-mer sequences are part of a 5 1-bp region which contains an imperfect dyad symmetry (Fig. 6). The differences between the double glnA and single glnL NR, binding sites could mean that NR, binds with a distinct conformation to the glnA and glnL regulatory regions. As shown in Fig. 3, these 19-mers are also present, at the same positions, in the S. typhimurium glnAglnL intergenic sequence. (d) Expression

of the glnL gene

A number of observations suggest that, under nitrogen-limiting conditions, the glnL protein level is lower than that of NR, even though both genes are translated from the glnALG transcript (Pahel et al., 1982; Goldie and Magasanik, 1982; Guterman et al., (1982). Post-transcriptional and translational control mechanisms have been proposed to explain the diminished synthesis of proteins encoded by internal genes in various E. coli operons (Barry et al., 1980; Dean and Nomura, 1980; Gold et al., 1981; Smiley et al., 1982; Gouy and Gautier, 1982). The lack of an efficient RB S was discarded since a recognizable AGGAG sequence (Fig. 2; Shine and Dalgamo, 1974) precedes the glnL gene. Also, there are

no si~~~cant stem and loop structures that could hide this RBS. The most stable one (at positions 566-615) has a theoretical dG of -5.7 kcal/mol. However, the possibility that a protein could stabilize one of these structures cannot be discarded. Another mechanism to maintain the glnL protein at low levels might depend on a high incidence of infrequently used codons. We are presently exploring this possibility.

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