J. Mo l . B iol. ( 1982)1 6 r. 5 1 9 5 3 1

Promoters of the malEFG and malK-lamB Operons in Escherichia coli Kl2 l . tlnsur,.cScHlrHrssst:H2fMat'nrcn Ho l'xuxc l H ucul:s Br.rnot'nr.r,n , a x n Ml H l tx R o s t:x n ER r;2 rUnité de Programmation Moléur,la,ireet Toricologie Génétique Institut Pasteur, 28 ru.edtL Dr Rour,75015 Po,r'is,France 2Laboratory of B'iochemistry,)ia,tional Cancer Institu,te National ltr,stitutesof Health, Be.thesrla. Md 20205. U.S.A. (Recei,ued 27 Mareh 1982. and i,n reuisedform 5 JuIy 1982) The nmll] rergion is composed of tu'o operons,rn,ultl'(l andmalK-lamB,transcribed divergentlyfrom a controlregionlocatedbetu'eenm.alllandm,alK.We determined the precisesitesat rvhichtranscriptionof the malB operonsstarts i,n ui,uo.These iriitiation sites define the promoter signals for mulEFG and malK-lamB. (-lomparison of the malE andm,alKpromotersequences revealshomologies that are likell' to correspondto the sitesat r.r'hichthe nrulT product and the c-'-clic-AilIP receptorprotein activateexpressionof the nrulBoperons.

1. Introduction The genes comprising the maltose system of Escheri,chiacoli, are located in two regions of the chromosome,malA and ma,IB (Fig. I ). The malA region is composed of the malT gene and the malPQ operon. The mn,lT product is the specific regulatory protein of the maltose system; malP and malQ encode maltodextrin phosphorylaseand amylomaltase,respectively(Hofnung et al.,l97l ; Debarbouillé et al., 1978:I)ebarbouillé & Schwartz, 1979).The m,alB region is composedof two operons, malUFG and m,alK-lamB, transcribed divergently from a control region located between mnIE and mnl,K. The products of the five genesof the malB region are involved in the transport of maltose and maltodextrins across the bacterial envelope. In addition, the lamB product is the cell surface receptor for several co l i p h a ges( lam bda, K1 0 ,. . .) (H o fn u n g , 1 9 7 4 ; R a i b aud et al ., 1979;S i l havy et al ., 1979). In the presenceof maltose, the malT gene-product (pmalT) activates expression of the three maltose operons. In turn, maIT transcription is positively regulated by cyclic-AMP receptor protein, CRP (Chapon,1982).Recent experiments(Bedouelle, unpublished results; Chapon. 1982) indicate that CRPf also acts directly on the t Present address: Biogen S.A..3 route de Troinex, 1227 Oarouge/Genève,Suisse. f Abbreviation used: []RP. c.r'clic-Al{P receptor protein. 519 0022-2836 I 82 l3205re-1 3 $03.00/0

O 1982 Academic Press Inc. (London) Ltd,

;-\20

molB

mo lA PmalT+ moltose

-

--

--==i--\

r

I mo lT

molP

jl molG

molQ

molF

molK

molE

IomB

_-'--'->

11 lt cAMP + CRP l,'lrt. | . ()rganizatiort ol tlrt' rrrirltost' svstenr an(l nlailr regulator\- ittteractiotts. Horizotital arrort's i p < l i r , i r t t ' t h t ' t l i n ' < . t i o l l so 1 ' t n r n s ( , r ' i p t i o n .\ - e r t i c r l â r r ( ) \ \ ' si n t l i t ' a t e u h i t ' h o l ) e r o l l s h a v t ' t h e i r t ' x l l r e s s i o t l ('RIt (ltlu'er r f i r . e t . t l r . r , r r r r t r o l l e lrllv t h t ' t t t r r l ' l ' p t ' o d t r ( . at l l ( l n l a l t o s e ( t r 1 r 1 r t 'l r) r r r t ) o r l r v t ' r ' t ' l i r ' - r \ ] l l ) a n t l r ) r r r t) .

exl)ression of the mel R operons whereas no (lirect effect of ('RP olt the expression of 'I'hus the maltose ol)erons àre tlll(ler complex m,alPQ has beert demonstrated. ('RP (strmmarize(l in Fig. I ). positive (.ontrol by both plnalT1 all(l \\te have previouslv reporte(l the DNA sequen(re of tl're etitire lnal IJ regtllatorv r e g i o n ( B e d o u e l l e & H o f n u n g . 1 9 8 2 ) . I n t h i s p a p e r . \ \ ' e ( ' h à r a ( : t e r i z et l i e 5 ' s t a r t s i t e s used in ?;iro fcrr transcription of the nlalEI'(I and ma,lK-lam'B operons. We dernonstrate that these sites {irnction l)rol)erlv tlllder rnaltose (rolltrol when introrluc,ed into (:ells on multitrollv lllasrnid derivatives. Positioning of the trariscription startsites of'these tu'o oltt't't)nsdefilles the ttt':.g=.1=11 =:=+{ za=*È:-==oi:::

Ë;;zi i:,tia:;;Êij

iÉ===;I::il1:-rzl r

t=z*;=rl?1tt+'=t

H . R I . ]I ) O T ' E I . T . E l i l '

AL Iogl

molE

3' -GAAGAAGCTAAAACCGCTCTC

GGCTCCGCCTITTGTAGTAGCAGCAATTACGCCTAlTACGCTCCTACGCACGTGGACAAAAATAAAAFTÂTTAGAlACC .-500

5'-GGACTCTAGTTTCTTTATACGGCAACCTCTTTCCATCCTCCTTGCCCCTACGCCCCACCGTCGCTTTG

ACTTTTGTGGATTTGCCTGAGATCAAAGAAATATGCCGTTGGAGAAAGGTAGGAGGAACGGGGATGCGGGGTGGCAGCGAAACACACTAGAGACAA ._400

^100 [à AATTGGCGGTAATGTGGAGATGCGCACATAAAATCGCCACGATTTTTGCAAGCAACATCACGAAATTCCTTACATGACCTCGGTTTAGTTCACAGAAC

TTAACCGCCATTACACCTCTACGCGTGTATTTTAGCGGTGCTAAAAACGTTCGTTGTAGTGCTTTAAGGAATGTACTG ' -3oo

aloo r o c . . \ r r l . . l a a dS. c i . .[ ' . , S . -.-7l 8 . i i t 2 i 5 6 . l l a r c h a l . ( ' . . ( i r e e n b l a t t . , l .& H o f i r u n g .l l ( l 9 7 t t ) ., 1 .B u c t e r i o lf.3 6 . l l 0 9 l l l { ) . l l c K e r t n t ' r ' . K . . S h i r n r r t t r k eF. I . ( ' o l t r t . I ) . . S c h r n e i s s n e rL. ' . . B r a c i t ' . ( ' . & I l o s e n b e r g .\ I . (l 981). In ( iette .1n pliJicutiott u ttrl A trul4.:is.r.ol. 2. ,l nulusi,sof )- ucleic ,1cids bu i l t r : t l n r u t i t,' l l t t h r r l s( ( ' h i r i k . j i a n . , J(.1 . & I ' a p a s .T . l i . . e t l s ) .p 1 r .l J 8 i 3 - - t 1 5I i.l s t ' v i t ' r / N < i r t h Hol ltrrrrl.A rrrst,t'r'dtr rn. ) l e s s i n g .. ] . . ( ' r ' t t r .R . & S e e b u r g .I ) . ( 1 1 ) 8 1 ). .\ ' u c l .- 1 c i d , r: ? c , y9.. : 1 0 93 2 1 . Miller, J. H. (1972).Erperinterûsin Molecular(lenetics.Cold Spring Harbor Laboratory',Neu, York. N l o n o d .J . . C o h e n - B a z i r e(.) . & C o h n . H . ( 1 9 5 1 ) .B i , o c h i mB. i o p h y s .A c t a . ? . 5 8 5 5 9 9 . . .R r r c t e r i o l .l g . i : 1 6 i | i . N e i d h a r d t . I " . ( ' . . I l l o t ' h . I ) . 1 , .r t S n r i t h . l ) . F ' . ( 1 9 7 . + ), 1 Q u e e t r .( ' . & R o s e n b e r gl.l . ( l { ) 8 1 ) .} ' r r c 1 .^ l c i d sr t c s . 9 . l l i l 6 . : r3 3 7 7 . I l a i l r a u d . O . . R o a . ) 1 . . I J r a u n - l J t ' e t o n( '.. c t S c h u ' a r t z .] 1 . ( 1 9 7 9 ) .l [ s l . ( l e r r .( ] e r t r t . 1 7 4 . 2 + l 2.1U. R a i b a n d . O . . l ) e b a r b o u i l l é ]. 1 . & ( ' o s s a r t .I ' } ( 1 9 8 2 ) ., 1 t t t t .M i c r r h i r t l . ( l t t . s t . P a s t e u . r ) , l . 3 3 A . .:r9 6.t. R o s t ' n b e r g).1 . & ( l o u r t . I ) . ( l { ) 7 9 ) ., f t r t t r rR. c r ' .( } e n c t . l 3 . 3 l 9 l } i r l l . S c h t ' r e ' (r i. F ' I i . . \ V t i l k i n s h a r vl l.. l ) . . A n r o t t . S . & l l o r r é . I ) . , J . ( 1 9 8 0 ) J. ' u c 1 . 1 c i d sf r e s . 8 . l]895 3907. S c , h t r t e i s s n e[ ''.r.. ( 1 o u r t . [ ) . . S h i r n a t a k t ' .H . & R , t . r s t ' n b e r] g l . ( 1 9 S 0 ) .I ' r o c .] - r r t .^ 1 r a d . , \ r i . . I r . , s . . { .7 7 . 3 l 9 t : ] 1 9 5 . S t : l r t t r e i s s t r e[ 'r. . ( ' o t t r t . I ) . . ] l c K e n n e r ' . K . & R o s e n l ) e r gi.\ I . ( 1 9 8 1 ) .] ' o t u r c ( L o n d o n ) . 2 9 2 . l7il- l 7,:r. S i e b e r r l i s [t '.. . S i r n p s o r rR. . I ] . c t ( l i l l r e r t .\ \ ' . ( 1 9 8 0 ) (. ' e | \ . 2 0 . 2 6 9- 2 8 1 . S i l h a v r 'T . . J . . I l r i c k r r t a nF. l . .B a s s f o r dI.) . . 1 . , 1 r(.' a s a c l a b a n . l l . , JS. .h u r n a n H . . A..Schu'artz. \ - . . ( i u a r e r r t e .L . . S c h u ' a . r t zl .l . c t B e c k u ' i t h ., L ( 1 9 i 9 ) . f l o l . ( l e t r .( k , n e t .1 7 4 . 2 1 9 2 5 g . [ ' l l t n a t t n . A . & I ) a n c h i n . A ( 1 9 8 2 ) .- l d r v n r .( ' u t l i c . t * u c l e o t i r lR e e s . 1 5 .i n t h e p r e s s . Edited b1t,1. H. Millcr