J. Mol. Biol. (1977) 111, 65--75

Nucleotide Sequence Changes Produced by Mutations in the lac Promoter of Escherichia coli

ROBERT C. DICKSON~, JOHN ABELSON, PETER JOHNSON

Department of Chemistry, University of California La Jolla, Calif. 92089, U.S.A.

Wrr,r S. REZNIKOFF AND WAYNE IV[. BARI~ES$

Department of Biochemistry, College of Agriculture and Life Sciences University of Wisconsin, Madison, Wisc. 53706, U.S.A.

(R~eived 27 July 1976, and in revised.form 3 December 1976)

Nucleotide changes occurring in class I mutants of the /ao promoter have been determined. Mutants L8, L37, L65 and L592 produce a G. C to T-A transition at nucleotide 19, while L29, L146 and L614 produce a C 'G to A.T transition at nucleotide 28. These two mutational sites are symmetrically located in a sequence in which 12 of 14 nucleotides are related by a dyad axis. This region may be the binding site for the catabolite gone activator protein. In a previous publication (Dickson etal., 1975) we presented the nucleotide changes seen in the class I I promoter mutants, L241 and L305, and the class TII promoter mutant, prla. Here we present the data which determined these nucleotide changes.

1. I n t r o d u c t i o n

RNA transcription is initiated at a region of DNA termed the promoter (Epstein & Beckwith, 1968). Genetic and biochemical evidence (Beckwith et al., 1972; de Crom- brugghe e ta l . , 1971; Eron & Block, ]971) indicate tha t the promoter for the /av operon o f Escherichia coli consists of a t least two functional units; these are the interaction sites for the catabolite gent act ivator protein and for R N A polymerase (Fig. 1).

The CAPw interaction site is defined (Fig. 1) as the region between the i-gone translation stop signal and the right end of deletion L1. This deletion and all point mutan ts tha t map within it are termed class I promoter mutan ts (Hopkins, 1974). Recent ly it has been shown tha t CAP binds specifically to an isolated restriction endonuclease-generated DNA fragment carrying t h e / a c control region but does not bind to the equivalent f ragment carrying the class I mutat ions L8 and L1 (Majors, 1975a). Phenotypically, class I mutan t s show as much as a 50-fold reduction in the

t Present address: Department of Biochemistry, University of Kentucky, Lexington, Ky 40506, U.S.A.

Present address: Department of Biochemistry, Washington University, St. Louis, Mo., U.S.A. w Abbreviations used: CAP, catabolite gone activator protein; UTP, uridine [,,-s2P]triphosphate;

ATP, adenosine [~-s2P]triphosphate; CTP, cytosine [~-a2P]triphosphate; GTP, guanosine [~.a2p] triphosphate.

5 65

66 R . C . D I C K S O N ET AL.

maximal inducible level of f~-galactosidase synthesis. The reduced level of ~ c expres- sion in these m u t a n t s is only slightly influenced b y the level of CAP and adenosine 3 ' ,5 ' -monophospha te (cyclic AMP) (Beekwith et al., 1972).

I n a previous publicat ion (Dickson et al., 1975) we noted t h a t in the proposed CAP site there is a region in which 14 of 16 nucleotide pairs are related symmetr ica l ly about a d y a d axis (Figs I and 2). We postula ted t h a t CAP binds to this symmetr ica l region and predicted t h a t class I point mu tan t s would occur within this region (nue]eo- tides 18 to 33 in Fig. 2). The results presented here suggest t h a t the CAP binding site m a y involve another symmetr ica l region (nucleotides 17 to 30 in Fig. 2) in which 12 of 14 nueleotides are related b y a d y a d axis.

There are two other classes o f m u t a n t s in the lao promoter . Class I I mu tan t s were predicted by Beckwith et al. (1972) and subsequent ly isolated by Hopkins (1974). Genetically, these m u t a n t s recombine a t a low frequency with deletion L1 and map just to the r ight of it in the R N A polymerase interact ion site. Phenotypieal ly , these mu tan t s show a reduct ion in the maximal inducible level of fl-galaetosidase synthesis as do class I mutants . However , unlike class I mutan t s , class I I mu tan t s are ve ry sensitive to the presence or absence o f CAP and cyclic AM~. For instance, class I I mu tan t s make from 10 to 30 times as much fl-galaetosidase in cells which are crp + cy a+ (a cell possessing CAP and adenyl cyclase, respectively) as t h e y do in cells which are crp- cya- . I n the same conditions class I mu tan t s make from one to seven t imes as much fl-galactosidase. Class I I mu tan t s are t hough t to affect the ini t iat ion of transcript ion. Recen t studies in vitro have shown t h a t L241 and L305 prevent for- mat ion o f the R N A polymerase-~zc P "open complex" (Reznikoff, 1976). I n the remalniug set of mutants , class I I I , the expression of the Zac operon in the absence of

I Promoter .I R.NA po!ymerase

6. col~ i Gene CAP site inTeract,on s~te Operator z Gene chromosome I I I I I

DNAsequence

I mRNA Glu Set Gly Gin slop fMi! Thr Met

GGJiJCCGGGCACIGiGCGCJ~CGC||r I A A | ~ [ G - ~ ? G * ~ [ ~ I ~ G G ~ A r 1 6 2 1 6 2 I rCJCJCIGGia JCJGC~iICaCCAIG CCTf ICGCCCC[CACTr TGCr162162 Jr162162162162162162162162 CCTTIGTCG|T&CICGI|r 5 s

I IO 20 30 40 50 60 70 80 90 I00 I10 120

--X plac 5 X8630

Delet ions F25a~ F ~ a W 227, L I

W225 SZO

)(8554 X8555--

I 1]" rrr l i e V v r S e g m e n t s i I I i I I I

FIG. I. The lac promoter-operator sequence. The LAC and CAL RNA sequences from Fig. 4 are presented as DNA sequences with the sequence extended 11 base-pairs to include the first 3 codons of the z gene (data from Maizels, 1973). Indicated above the sequence are the proposed locations of the i-gene, p (CAP and RNA polymerase sites), o and z genes. The regions of symmetry in the CAP site and o are shown as is the mRNA start site which has been determined by Maizels (1973) and Majors (19755). Below the sequence, the approximate locations of the deletions used in the RNA hybridization procedures are shown as well as the segments defined by some of these deletions. The numbers below the DNA sequence indicate individual nueleotide pairs starting wi~a the first nucleotide beyond the lac i gene stop codon. Dots between the two strands of DNA indicate dyad axes and the lines above and below the strands indicate the nucleotides which are elements of the dyad axis.

N U C L E O T I D E C H A N G E S I N lac P R O M O T E R 67

Protected by represser

5 lUlILKG~UI|IASCV,~IIC~UI'TA||ITUtT ~|CJLCTC|||IL||C/Cr162 ~|II|IfA|yI$|UeC~|A~il/TTCACACAC r~ILAC&GCTIIGIr r 'TT|IICIiI|TUC~r162 I'TIr | C I l l | ' Ii|'ICr l ' t r ' l ' ' t 'C 'G ' r CU|C' ' ' r ' C . . . . . ...TIIC,CIr162 1'r162 5 '

"-...

. / " I IO 20 50 40 50 60 70 80 90 ....... I00 llO 120

/ The promoter "-....

:" "%. / "...

.." Hk:Jh G'C HkJh A'T High G'C .........

~CGC~CGC~rr~TOAOT~%CTCACTCArrA'OGCACCCCA~C"TTacACrTTM~cr~coocTC~rTGTOTOOii CG CG TTG CGT TAAT TACAC T CAATC.GAG TG AGTA ATC CGTG G GGTCCG AAATGTG AA~'ACGAAG G CCGA G CATACAAC ACAC C T

t I L I

T A RepeatincJ T pentomer T

F25a, F~6a deletion

LI, W227 deletion

FzG. 2. The lac promoter sequence. The detailed sequence of the lac promoter shows the location of the various promoter mutations and interesting structural features such as the CTTTA repeats. the CAP symmetry elements, and the high G. C and A. T blocks. The class I [ mutation L305 deletes a G. C base-pair. The sequence protected by the represser was determined by Gilbert & Maxam (1973). The mRNA start site was determined by Maizels (1973) and Majors (1975b).

CAP or cyclic A M P is e l eva t ed to levels above t h a t found in t h e wi ld - type . These m u t a n t s p r e s u m a b l y p r o m o t e t h e i n t e r ac t ion of R N A po lymerase w i th t he /ac p romote r . W e have g iven resul ts (Dickson et al., 1975) for nuc leo t ide changes in two class I I m u t a n t s , L241 a n d L305, a n d for t he class I I I m u t a n t , p r l a (Fig. 2). I n th is

p a p e r we p re sen t d a t a suppor t i ng these nuc leo t ide changes.

2. M a t e r i a l s a n d M e t h o d s

(a) Bacterial and phage strains

Characteristics of the class I mutan t s L8, L29 and L37 are described b y Beckwith et al. (1972), while L65, L82, L83, L146, L208, L271, L408, L450, L592 and L614 are described by Hopkins (1974). Class I I mutan t s L157, L241, and L305 are described b y Hopkins (1974). Class I I I mutants , including p r l a are described by Ardi t t i et al. (1973). These bacter ia l mutan t s were t ransferred to specialized l ambda or r t ransducing phage as described b y Barnes et al. (1974). The phage were grown and purified by the procedures of Reznikoff et aL (1974).

(b) Experimental approach for determining the nucleotide change in point mutants

Nucleotide sequence changes caused b y point mutan t s in the lac promoter were deter . mined b y analyzing R N A transcr ipts of the lac promoter region. Techniques for syn- thesizing s2p-labeled R N A in vitro, for isolating R N A using the 2-step R N A - D N A hybr idizat ion procedure and other techniques used in this work have been presented in detai l by Barnes et al. (1975).

(e) 17omenelat~re

R N A transcr ibed from the promoter region in the same direction as lac m R N A has the same nucleotide sequence as the top s t rand in Fig. 1 and we refer to this as LAC RNA. CAL R N A refers to R N A transcr ibed in the opposite direction. LAC and CAL R N A were t ranscr ibed from ~ or r DNA templates , respectively.

5*

68 R . C . DICKSON E T A.5.

3. Results (a) IVudeotide Jtanges in class ! promoter mutants

LAC RNA labeled with UTP was transcribed from a template carrying the mutant L8. The two-dimensional ribonuclease A oligonucleotide map of this RNA (Fig. 3) contains an oligonucleotide, labeled a, which is not present in wild-type RNA. The mobility of this oligonucleotide suggests a composition of A2GU. Oligonucleotide a is not labeled by CTP so its 3' nucleotide has to be U and its nearest neighbor cannot be C. Ribonuclease T1 digestion of UTP-labeled oligonucleotide a yields two labeled

12A

a

2B

12A

9

10+I I

J

Wild~ type LB

Fro. 3. Autoradiograph of a 2-dimensional ribonueleaso A oligonuelootido map of LAC RNA complementary to DNA carrying the mutan t LB. For details see Results, section (a).

products, AAG and U, establishing that the sequence of the oligonucleotide is AAGU and tha t its nearest neighbor is U. Similar analyses using ATP and GTP-labeled RNA support this conclusion. For unknown reasons the intensity of oligonueleotides 9 and 10~-11 was variable. The only other nucleotide change we could detect in the L8 RNA shown in Figure 3 was in oligonucleotide P16. In wild-type RNA there are two products, GAGU(U) and GGAU(A), that co-migrate as oligonucleotide P16 (see Fig. 4). However, only GGAU(A) is present in UTP-labeled L8 RNA since digestion of oligonucleotide P16 with ribonuclease T 1 gives only AU. I f P16 contained GAGU(U) then the products AG and U should be present also. These data suggest tha t nucleotide 19, representing the 5'G of GAGU, has changed to an A to give the new ribonuclease A product AAGU(U). I f this is true, there should be a new product

i Gene

N U C L E O T I D E C H A N G E S I N lac P R O M O T E R 69

LAC strand PIS PI5 PI2 Pit P4A P7 PI6B P6 PIOA PtOB

T6 1"2 T4BTSTST2 T5 Tt8 TST3TI48 T 19 T7 TI7 5' GG~-A-'~'-~GG~'A"~"~'-~--'~AAC~AAUUAAUG'U"~J~-~OUAGCU C A C U C A U U A G G ~ G ~ : U U U A C A C U U U A U G '

I I

I I I I I I I 3 ~ GCGC GU CACUCGCGUU GCGUUAAUUACAC UCAAUCGAGUGAGU AAUCCGUGGGGUCG GAAAU GUGAAA UAC

T5 TI9B T2TI7 T2 T25 T4T9T4 TI9A T9 TI2 TI5 1"9 T I6 ~ t I I I I , , ~ J I . I I I I I - -

PTA P48 P9B PII P7B PI8 PI2 PIOA

CAL stran d

LAC strand

P 9 PI7 PI6A P4B P7 PI4

T I7 TI6 TIO TI4A TI3 TBT8 TI5 TST51"2 T20 T4A CA C U U U A U ~ UUC C ~ G ~ - G ' ~ = G ' ~ - ~ - ~ - ~ G ~ - ~ ' ~ - G ~'~ G~U A AC A AU UU C A CA C AG'G ~-~''~A 3'

e'o T'o 8'o 9'0 ,~o I~o ,~o I I I I I I I

G LJ ~;AAA U ACi G A ~ G ,G CC.G A.G C A U ACA(A~C Ace UU AA C A C UCjp C C UA U L~p UUAAA,~_~ G~p UC C UU q ~ 5' T9 TI6 T7 T3 T4 T26 T21 T20 TgT9 T25 TIO

!

LPIOA ~ PI4 P9A P4A P7A PIOB

CAL strand

Z

Gene

FrG. 4. LAC and CAL promoter-operator sequences. The LAC and CAL promoter-operator sequences are presented with significant T1 oligonucleotides (labeled T) and ribonuclease A oligonucleotides (labeled P). The numbered ollgonucleotides refer to these shown in Figs 3, 5, 6, 7, 8.

UAAG(U), representing nucleotides 18 to 21, in a two-dimensional ribonuelease Tz oligonueleotide map of L8 RNA. This prediction was verified as follows. L8 RNA labeled with ATP, U T P or GTP shows a new oligonucleotide in a two-dimensional ribonuclease T1 oligonucleotide map (data not shown). I ts position suggests a compo- sition of UA2G. After ribonuelease A digestion of the new UTP-labeled RNA one product, AAG, is obtained, while ATP-labeled RNA gives U and AAG. Thus the sequence of this new oligonucleotide is UAAG and its nearest neighbor is U. These data show tha t muta t ion L8 is a transition muta t ion of G- C to A. T at nueleotide 19 as shown in Figure 2. The two-dimensional ribonuclease A and T1 oligonucleotide maps of RNA made from the mutants L37, L65 and L592 were the same as L8 indi- cating tha t these mutan ts are identical to L8.

LAC RNA transcribed from a template containing mutan t L614 was labeled with GTP. The two-dimensional ribonuclease T1 oligonucleotide map (Fig. 5) lacked oli- gonucleotide 19 but contained a new oligonucleotide, b, not found in wild-type RNA. The mobil i ty of b suggests tha t it is derived from oligonucleotide 19 but contains an extra U residue. This idea was supported by initial studies in which UTP-labeled oligonucIeotide b was digested with ribonuelease A. The products AC, AU, C and U were obtained. The first three products are normally obtained from UTP-labeled oligonueleotide 19. The extra U indicates tha t there is a Py r U U sequence in oligo- nucleotide b. Digestion of UTP-labeled 19, whose sequence is CUCACUCAUUAG(G), with ribonuclease U2, gives one mole of UUA and 2 moles of CUCA. The 3' neares t neighbor of one CUCA is U and the other is C. Similar digestion of UTP-labeled oligonueleotide b gives the two usual products UUA and CUCA, plus a third product which moves more slowly than UUA on DEAE paper in 7 % formic acid. Digestion of the third product with alkali gives equal amounts of C and U suggesting tha t its sequence is CUUA. Alkali digestion of the CUCA gives equal amounts of C and A so its nearest neighbor is U. Tha t the 3' nearest neighbor of CUUA is C was verified by showing tha t this product is present in CTP-labeled RNA. RNAase A digestion of

?0 R . C . DICKSON E T A L .

'!r i .,', 13

L ;

16

i 10

l

13

16 14

Wild-type L614

FIGo 5. Autoradiograph of a 2-dimensional ribonuclease T1 oligonucleotide map of LAC RNA complementary to ])NA carrying the mutant L614. For details, see Results, section (a).

CTP-labeled RNA gave equal amounts of AC and U. This result precluded the possi- bility tha t a U had been inserted between nucleotides 30 and 31 or 31 and 32. Verifica- t ion was obtained by showing tha t a T1 digest of GTP-labeled CAL RNA synthesized from mutan t L29 gave a new T1 product UAAG (data not shown), which would be complementary to CUUA but not to CUUCA. These data establish tha t the ribo- nuclease U2 product CUCA(C) obtained from oligonucleotide 19 has changed in mutan t L614 to CUUA(C) so the sequence of oligonucleotide b is CU_UACUCAUUAG, where the underlined nucleotide represents the C .G to T . A transit ion caused by mu tan t L614 at nucleotide 28 (Figs 2 and 4). The mutants L29 and L146 give the same nucleotide change as L614 (data not shown).

(b) Nucleotide changes in class I I promoter mutants

GTP-labeled CAL RlqA was transcribed from a template carrying the mutan ts L241. A two-dimensional ribonuclease T 1 oligonucleotide map of this RI~A (Fig. 6) contains an oligonucleotide, c, t ha t is not present in wild-type and it lacks oligo- nucleotide T15 [UAAAG(C)]. The mobili ty of c suggested tha t it was derived from oligonucleotide T15 by changing an A to a U. This was verified by digesting G- labeled oligonucleotide c with ribonuclease A. Only the product AAG was obtained,

N U C L E O T I D E C H A N G E S I N lac P R O M O T E R 71

O �9

Witd-type L241

FIo. 6. Autoradiograph of a 2-dimensional ribonuclease T1 oligonucleotide map of CAL RNA complementary to DNA carrying the mutan t L241. The ends of this RNA are defined by deletions X8554 and W227. For more details, see Results, section (b).

suggesting that the sequence of c is UUAAG which would mean that nucleotide 51 (Fig. 2) has been changed from T-A to A.T.

Further verification of this change came from a two-dimensional ribonuclease A oligonucleotide map of L241 CAL RNA. As expected, such a map lacked oligo- nucleotide P12 [AAAGC] and it contained a new oligonueleotide AAGC (data not shown).

ATP-labeled OAL RNA was transcribed from a template carrying the mutant L305 and a two-dimensional ribonuelease T1 oligonueleotide map was prepared. This RNA lacked oligonueleotide T15 (Fig. 7) but it contained a new oligonucleotide, d, tha t had a mobihty similar to T19A and T19B (Fig. 4). To facihtate the sequence analysis, oligonueleotides T19A and T19B (see Fig. 4) were removed by using DNA carrying deletion W227 for the second I~NA-DNA hybridization of the RNA purifi- cation procedure. The T1 ohgonucleotide map of GTP-labeled L305 RNA lacked both oligonucleotide T15 [UAAAG(C)] and T12 [CCUG(G)]. In the CAL strand sequence T12 is the 3' neighbor of T15 (see Fig. 4). These data suggested that oligonueleotide d was derived from oligonucleotides T15 and T12. Oligonucleotide d, labeled with CTP, was digested with alkali and two products, A and C, were found in equal amounts. This indicated that d was derived from T15 and T12 by a deletion of the 3' G in T15.

Ribonuclease A digestion of ATP-labeled d gave A/kAC and U, while UTP-labeled d gave only 0, and GTP-labeled d gave U and G. These data show that the sequence of oligonucleotide d is UAAACCUG(G). We conclude that nucleotide 48, a G, h a s been deleted in mutant L305 resulting in the fusion of oligonucleotides T12 and T15.

72 R . C . DICKSON E T A L .

�9 - . . ' 5 r

� 9 . - : r

Wild -- type L305

Fro. 7. Autoradiograph of a 2-dimensional ribonuclease T: oligonucleotide map of CAL RNA complementary to DNA carrying the mutant L305. The ends of this RNA are defined by deletions X8554 and W227. For more details, see Results, section (b).

Fur ther proof of this was obtained from a two-dimensional ribonuclease A oligonucleo- tide map of C-labeled L305 CAL RNA (data not shown) which lacked oligonucleotide P12 [AAAGC(C)] (refer to Fig. 4) but contained a new product, AAAC. The G in oligonucleotide P12 is nucleotide 48 and its deletion generates the AAAC sequence.

(c) Nucleotide change in a class I I I promoter mutant

We examined the oligonucleotide composition of RNA transcribed from several class I I I mutants including pr la , 2c, 12A, 13A, 19 and L8-UV5. All of these RNAs had reduced amounts of LAC oligonucleotides TI6, T10, T14A and TI3 and also CAL oligonueleotides T3 and T26 (see Fig. 4). Only one mutan t showed a discernable nucleotide change. As shown in Figure 8, a two-dimensional ribonuclease Tz oligo- nucleotide map of G-labeled CAL RNA transcribed from a template carrying mu tan t p r l a lacks ohgonucleotide T3 [CCG(G)] and it contains a new oligonueleotide, e. Digestion of GTP-]abeled e with ribonuclease A gives equal amounts of G and C, indicating tha t the 3' end of oligonucleotide e is CG(G). The 3' end of oligonucleotide T3 is also CG(G) suggesting tha t e is derived from T3. The mobil i ty of e indicates tha t it also contains one U, at least one A and two Cs or two As and one C. The nueleotide

N U C L E O T I D E C H A N G E S I N l a c P R O M O T E R

. ~.~.~:~.~ "~.,. : . ~ - : . .. - �9 . . . . ~ . : / ';, . , , - : . ~ : . ~ . - ' : . ~ ,

' . . . " ' - . ' " - . . ' ~. . , - . . . ' - k " " �9 : , - : ~ - : " - : : k ~

' e t ~ - " , : : ( " ...... :.7 q l e ' . ;:~;k... ..:~-% ~ . . . . = ~,. " T : . . " Y ~ ' ' " ' ~ ~ . . : . . . . . , , : . . . . ", , . : " . . ' . o : '~ :~ -? - * .~]i':,/

. �9 , ~ , �9 . , , . ..-'..,-..::~%. . . . . : ~ : " " " " , . . . . ' ' ' " ( A - - : . .:.',,k*..~

�9 ' . �9 , . , - - I . - . " . . r_ ' ; '~ L " :".::': '~:~.

, . , , . "* ~ " ~ ' ' - ~ F . ' ~ : ' , ~ , , ~ "

�9 ~ . . . . . I , : . " - . . 1 ~ ' _ . ' ~ ' ~ . ' ! .

" " ' T ~ : ": . . . . ~ . . . . " " : " " ~ ! " " " - " ~

. . . .

i~ ' : " - ... t

. : . : -- : , : :

-:'d ," ~ :>!'~ ~~- '~ :~ i

7 3

t

. .

Wild -- type pr lo

F I o . 8. A u t o r a d i o g r a p h o f a 2 - d i m e n s i o n a l r i b o n u c l e a s e T 1 o l i g o n u c l e o t i d e m a p o f C A L R N A

c o m p l e m e n t a r y t o D N A c a r r y i n g t h e m u t a n t p r l a . F o r d e t a i l s , s e e R e s u l t s , s e c t i o n (o) .

sequence around oligonucleotide CAL T3 is 5' AGCCGG 3'. These data are only compatible with mutant p~la being a transversion of nueleotide 69 from G to T as shown in Figure 2. This would yield a new RNA product, AUCCG(G), which would have a mobility like oligonucleotide e and would give equal amounts of G and O after digestion of G-labeled material with ribonuelease A. Because of technical difficulties in obtaining high yields of RNA with this mutant, the sequence change was not verified further.

74 R . C . D I C K S O N E T A L .

4. Discussion We have examined nucleotide changes in three classes of promoter mutants in

order to understand the relationship between the nucleotide sequence and the function of the /ac promoter. In our initial sequence studies (Dickson et al., 1975) we noted that in the approximately 35 base-pairs comprising the CAP interaction site, the region between the translation stop codon for the i-gene and the right end of deletion L1 (Fig. 1), there is a nucleotide sequence in which 14 of 16 nucleotide pairs are related by a dyad axis (these nucleotides, 19 to 33, are underlined in Figs 1 and 2). We suggested that these nucleotides comprised the CAP binding site and predicted tha t class I promoter mutants would occur in this region. There are two reasons for postulating tha t nucleotides 19 to 33 might be the CAP binding site. First, the likeli- hood of a random occurrence of so highly a symmetrical site is low (Dykes et al., 1975) and its occurrence therefore suggests tha t it has some specific function. The second reason follows from the structure of CAP. Since CAP is a dimer composed of two identical 22,000 molecular weight monomers (Riggs et al., 1971) it could have a dyad axis and therefore bind to nucleotide pairs also related by such a symmetry element. I f the interaction between CAP and its binding site involves symmetrical contacts then one might expect mutations in the binding site to be symmetrical also (Gilbert & Maxam, 1973). However, the mutations L8 and L614 are not symmetric- ally arranged around the dyad axis located between nucleotides 25 and 26 (Fig. 2) and involving nucleotides 19 to 33. There are two possible explanations for this. CAP may bind symmetrically to a site different from the one involving nucleotides 19 to 33. For instance, as noted by Dykes et al. (1975) nucleotides 17 to 30 form a dyad axis in which 12 of 14 nucleotides are symmetrical about an axis located between nucleotidcs 23 and 24 as indicated in Figures 1 and 2. Mutations L8 and L614 are also symmetrically related around this axis with L8 being five nucleotides to the left and L614 being five nucleotides to the right of the center of symmetry. Thus CAP might interact symmetrically with this region, nucleotides 17 to 30, rather than with nucleotides 19 to 33 (Figs 1 and 2). Alternatively, as in the repressor-operator inter- action (Gilbert et al., 1975), CAP binding to nucleotides 19 to 33 may involve sym- metrical contacts but these may not wholly govern the overall interaction, so that CAP may interact more strongly, and thus non-symmetrically, with one half of the symmetry element.

From genetic evidence Miller et al. (1968) suggested that L8 and L37 map at the same site. Our data confirm this and show that two other mutants L65 and L592 also occur at the same site as LB. Similarly, mutants L614, L29 and L146 produce identical nucleotide changes. Such repeated selection for the same two sites might result from genetic "hot spots" or it could mean that nucleotides 19 and 28 form the most important contacts with CAP.

The class I I promoter mutants, L305 and L241, precede the mRNA initiation site by about 35 base-pairs or 120 A. These mutations are not in the DNA fragment pro- tected from DNAase digestion by RNA polymerase when it is bound to the initiation site (J. Gralla, personal communication). Currently there are two models which a t tempt to explain the effects of these mutants. One model envisages RN/k poly- merase interacting first with the promoter about 35 base-pairs before the mRNA initiation site (Blattner et al., 1972). Another model envisages polymerase binding at the mRNA initiation site with other contacts forming, such as at the site of L241 or L305. In both models RNA polymerase could make contact with GAP.

N U C L E O T I D E CHANGES IN lac PROMOTER 75

The class III mutan t p r l a precedes the transcription initiation site b y 16 base-pairs. I t is not within the promoter homology region, nucleotides 73 to 79 in Figures 1 and 2, first observed by Pribnow (1975), indicating tha t bases outside this region are also important for promoter function.

In considering the possible consequences of deletion mutat ions such as L305, and also insertions, it occurred to us tha t such mutat ions could exert an effect in two ways. First, as with transition or transversion mutations, deletion or insertions could change a nucleotide-to-protein contact. Secondly, the nucleotide removed by a deletion or the nucleotides adjacent to an insertion might not normally make contact with a protein. However, if such nucleotides were si tuated among the ones involved in protein contacts then the effect of the muta t ion on the nucleotides involved in con- tacts would be to change the distance between them by 3.4 A and rota te them 36 ~ out of phase. Likewise, two adjacent and interacting proteins bound to their respec- t ive nucleotide binding sites would be ro ta ted out of phase and the distance between them would be changed as a result of an insertion or deletion occurring in the nucleo- tides between the two protein nueleotide binding sites.

This work was supported by National Institutes of Health (NIH) grants 1-1%01-GM- 19670 and CA 10984, by National Science Foundation grant GB-20462, by the Cancer Research Coordinating Committee of the University of California, and by the Wisconsin Alumni Research Foundation. One of us (J. A.) was supported by a faculty research award from the American Cancer Society. Another author (W. S. 1%.) was supported by a career development award S-K04-GM-30970 from NIH, and a third author (W. M. B.) was supported by NIH training grant GM-00236.

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