J. Mol. Biol. (1980) 139, 551-556

LETTERS TO THE EDITOR

lac Promoter Mutation P’115 Generates a New Transcription Initiation Point

The Eecherichiu coli luc P’115 mutation changes the base-pair where in vitro RNA synthesis initiates from an A/T to a T/A (Maquat et al., 1980). Such an alt,eration results in a catabolite gene activator protein-independent phenotype (Reznikoff, 1976; Maquat et al., 1980). In the absence of catabolite gene activator protein and CAMP, the altered base-pair of the mutant DNA is not used in vitro to initiate messenger RNA, but RNA synthesis is initiated at a new site estimated to be 12 base-pairs downstream (+ 13) from the P + transcription initiation site ( + 1). In the presence of the positive affecters, lacPrl15-directed transcription initiates near the + 1 site. Thus, CAMP-activated catabolite gene activator protein at least in part overcomes the effect that the P’llS base-pair alteration has on the stitrt site of RNA synthesis. The ZacPrl 15 studies suggest that catabolite gene act&&or protein stimulates transcription initiation only at discrete locstions (at) or near + I but, not at or near + 13).

Pr (class III) luc promoter mutations are sequence changes which enhance the expression of the lac operon in the absence of the positive aficctors: the catabolite gene activator protein and adenosine 3’, 5’-cyclic phosphate. The mutant promoters are tfhought to have an increased affinity for RNA polymerase relative to the wild-type promoter such that the binary RNA polymerase-promoter complex forms at, a faster rate and transcription initiation occurs more frequently (Reznikoff, 1976; Maquat & Reznikoff, 1978). This has been found to be the case for ail P’ mutations analyzed to date. Sequence analysis has indicated that the lacP’115 mutation is an A/T + T/A transversion at the +l site (Maquat et al.: 1980; see Fig. l), t,he major start site of ilz vitro Zac messenger RNA synthesis (Maizels, 1973; Majors, 1975; Maquat & Rezni- koff. 1978). It is of interest t,o determine how an alteration of the base-pair which codes for the first mRNA nucleotide results in a CAP?-CAMP-independent phenotype. We describe experiments utilizing an in vitro t,ranscriptional system which analyzes the funct,ional interaction between RNA polymerase and ZucPp115 DNA.

To approximat,r t’he start site of in vitro ZucP’115 RNA synthesis, 2acPrl15- containing restriction fragments were used as templates in the transcriptional assay. The mobility of the major transcripts produced by Hue111 203 kzcP+ corresponds to RNA molecules which are 63 and 64 bases (Maquat & Reznikoff, 1978; Maquat et al.. 1980; see Fig. 2). This is true whether or not CAP and CAMP are present. These are the sizes expected if initiation is at the +l and -1 sites, and if RNA polymerase runs to the end of the fragments (Maizels, 1973; Majors, 1975: Maquat & Reznikoff, 1978). From fingerprint and/or size analyses, all previously tested lacPp templates produce transcripts of sizes which correspond to initiation at the fl and -1 sites (Maquat & Reznikoff, 1978). HaeIII 203 ZucP’115, in the absence of CAP and CAMP, does not direct the synthesis of 63 to 64-base RNA but rather directs the

t Abbreviation used: CAP, cetabolite gene activator protein.

551

0022-2836/80/150551-06 $02.00/O 0 1980 Academic Press Inc. (London) Ltd.

552 L. E. MAQUAT AND W. S. REZNIKOFF

(0 1 -40 -30 -20 -10 +I +10

. . . . . .

/ocP+ A-C-C-C-C-A~GC-T-T-T-A-C-PrC-T-T-T-A-T~-C-T-T-C-C~G~-T-C-~T-A-T-G-T-T~-T-~T-GG-A-A-T-T-G-T-G-~ __- -___-

(b) -40 /

J .

-30 -20 -10 +io

. . .

J +I

. .

/ucP’ I I 5 A-C-A-C-T-T-T-A-TG-C-T-T-C-CGGC-T-C-G-TG-T-A-T-G-T-T-G-T-G-~G~-~-T-G-~-A-~~-A~-A-A~-A-A-T-T-~ --

FIQ. 1. The lacP+ and Pr115 promoter sequences. (a) The lacP+ sequence and the location of the P’115 mutation (Maquat et al., 1980) are depicted. Base-pairs are numbered relative to the site of in vitro Zac mRNA transcription initiation (Maizels, 1973) which is defined as +- 1. (b) The ZacP’115 sequence is displaced from the P + sequence by 12 base-pairs so that the start points are aligned. Similarities with conserved sequences described by Rosenberg & Court (1979) are under- lined.

synthesis of an estimated 51-base RNA (Fig. 2, track (c)). Transcription off of Ah 95 ZucP’115 was also compared to transcription off of Ah 95 ZucP UV5. The 95 base-pair Ah fragment should direct the synthesis of a 36 or 37-base RNA if initiation were to take place at the +l or -1 site (Maquat $ Reznikoff, 1978). This is what the ZucP UV5 template does (Fig. 2, track (f)). I n contrast, Ah 95 lacPrl15 direcbs the synthesis of a smaller, approximately 24-base RNA (Fig. 2, track (g)). These results indicate that P’115 does not initiate transcription at the $1 and -1 sites, but initiates transcription approximately 12 base-pairs downstream from these sites.

A possible rationale for the existence of the new CAP-CAMP-independent mRNA start site in lacP’115 can be deduced from an analysis of the relevant DNA sequences (Fig. 1). There are two regions of high sequence homology among prokaryotic pro- moters which have been proposed to function in RNA polymerase binding. One is a heptamer (the so-called Pribnow box) centered about ten base-pairs upstream :from the site of transcription initiation (Pribnow, 1975; Schaller et al., 1975; Reznikoff & Abelson, 1978; Scherer et al., 1978; Rosenberg & Court, 1979). In changing the P+ +l site from an A/T to a T/A, P’115 generates a new Pribnow box sequence

of the AZuI 95 base-pair fragment was preincubated for 3 min at 37°C in the presence or absence of 0.25 pg CAP and 1 miw-CAMP. RNA polymerase was then added at a 2.7 : 1 molar ratio of enzyme to DNA and the incubation was continued. After 30 min, 1 ~1 of a 2 mg/ml solution of heparin was added. One minute later, the reaction was made 200 pM in ATP and GTP, 10 PM in CTP and 7 ,UM in [c~-~~P]UTP. RNA synthesis was terminated after 10 min by the addition of 100 ~1 of ice-cold transcription buffer. Transfer RNA (100 pg) was added and the mixture was extracted with phenol. The RNA was precipitated with ethanol, washed, dried in txzcuo and dissolved in 16 pl of 0.1 x Tris-borete/EDTA electrophoresis buffer (Maquat & Reznikoff, 1978) and 4 ~1 of 60% (v/v) glycerol, 0.25% (w/v) b romophenol blue and 0.25% (w/v) xylene cyanol. The transcripts were electrophoresed through a 10% ( w v acrylamide/‘lM-urea slab gel and the gel was auto- / ) radiographed. (a) Hoe111 203 lacP+ without CAP and CAMP; (b) Hoe111 203 ZacP+ with CAP and CAMP; (c) Has111 203 ZacPr115 without CAP and CAMP; (d) Hoe111 203 ZacP’llB with CAP and CAMP; (e) Hoe111 203 hcP LSUV5 without CAP and CAMP; (f) AEuI 95 ZacP LSUV5 without CAP and CAMP; (g) AZuI 95 ZacPr116 without CAP and CAMP. 0, origin; XC, xylene cyanol; BPB, bromphenol blue.

xc-

BPB-

LETTERS TO THE EDITOR

la) lb) (d)

63 - 64

51 ba ses

36 -

24 bc IS%S

FIG. 2. Autoradiograph showing the transcripts synthesized off of &P-containing restriction fragments. Reactions (Maquat & Reznikoff, 1978) were done in a 20 pl volume consisting of 30 mu- Tris.HCl (pH 7.9), 0.1 mhr-EDTA, 3 mM-MgCl,, 100 mix-KCl, 0.1 mM-dithiothreitol, 0.5 mg bovine serum albumin/ml. 0.1 @ of the HaelI1 203 base-pair fragment or a molar equivalent

554 L. E. MAQUAT AND W. S. REZNIKOFF

(A.-A-T-T-G-T-G + T-A-T-T-G-T-G) in which five of seven positions including the three most conserved positionslunderlined above) are present. Approximately ten base-pairs downstream from the center of this hepta,mer is the proposed +13 start site for P’115. The second region of high sequence homology among promoters is

usually centered near -35 (Gilbert, 1976; Seeburg et al., 1977; Reznikoff & Abelson,

1978; Scherer et al., 1978; Rosenberg & Court, 1979). As shown in Figure 1, there are some sequences between 26 and 40 base-pairs upstream from the +13 site which resemble this conserved region and may function in the RNA polymerase-promoter interaction. Alternatively, the P + -35 sequences may be the functionally signi- ficant region of this promoter. This would imply that a flexibility exists in the distance between the functional “-35 region” and the Pribnow box.

It has been shown (Majors, 1975; Reznikoff, 1976; Maquat & Reznikoff, 1978) that the phenotypic properties of most ZacP mutants are reflected in the in vitro RNA polymerase binding properties of their ZacP DNAs. That is, Pr mutant DNAs bind RNA polymerase and form a functional complex faster than P+ DNA, and most, P- mutant DNAs fail to bind RNA polymerase with any appreciable efficiency. The results of transcriptional studies designed to measure the in vitro rate of functional enzyme-ZacP’115 complex formation are shown in Figure 3. The yield of functional

Incubation time (mid

Fro. 3. Progress of reaction curves for RNA polymerase binding to Hoe111 203 ZacP’115 and Hue111 203 ZacP+ in the absence of CAP and CAMP as determined by the transcriptional assay. 0.1 pg of Hue111 ZaeP-containing fragment was preincubated for 3 min at 37°C in the presence or absence of 0.25 pg CAP and 1 mM-CAkIP. RNA polymerase was then added at a 2.7 : 1 molar ratio (10 : 1 weight ratio) of enzyme to DNA and the incubation was continued. After varying periods of time, 1 ~1 of a 2 mg/ml solution of heparin was added. One minute later, the reaction was made 200 PM in ATP and GTP, 10 p~ in CTP and 7 p in [a-3aP]UTP. RNA synthesis was ter- minated after 10 min by the addition of 100 ~1 of ice-cold transcription buffer. Reaction conditions, RNA preparation for electrophoresis, and electrophoresis were described by Maquat t Reznikoff (1978) and are outlined in the legend to Fig. 2. Autoradiographs were made of the gels, and the RNAs corresponding to full-length transcripts of the Hue111 fragments were excised and quanti- tated by CQrenkov radiation analysis. Times of incubation with RNA polymerase prior to heparin addition are plotted aerau8 counts incorporated into full-length transcripts. The counts incor- porated value for P’115 expression was multiplied by 15/12 to compensate for the fact that the 51-base transcript contains only 12 U residues as opposed to 15 in the 64.base transcript. -O--O--, Hoe111 203 ZacPP115 without CAP and CAMP; -A-A---, Hoe111 203 hcP+ without CAP and CAMP.

LETTERS TO THE EDITOR 555

complexes at later time points (20 and 30 min) does not differ significantly between the Pp115 and the P+ template. However, at short preincubation times (< 2 min)

the P’115 template yields 1.5 to 2 times as much full-length transcript as the P+ template indicating that, in the absence of CAP and CAMP, Hue111 203 lacP’115 forms a functional complex with RNA polymerase faster than does Hue111 203 lhP+.

In the presence of CAP and CAMP, the predominant RNA species synthesized off of Hue111 203 lacPr115 is estimated to be 62 bases (Fig. 2, track (d)). Thus,

CAMP-activated CAP stimulates RNA chain initiation near the $1 site and fails to stimulate initiation at or near the +13 site. By restricting the site of initiation to near $-1, CAP at least in part overcomes the effect of the P’115 base-pair alteration in changing the conformation and/or the positioning of RNA polymerase. The positive transcriptional affector may block this effect of the Pr115 base-pair change because of physical constraints in the mechanism of CAP-mediated transcription activation. The results obtained with lacP’115 do not determine if CAP facilitates the formation of a functional RNA polymerase-lacP complex by enhancing localized DNA helix destabilization (Dickson et al., 1975) or by a direct protein-protein interaction (Gilbert, 1976). These results do suggest that CAP can promote the initiation event in only a

limited region of the promoter.

WC thank Alan Kinnihurgh for critical reading of the manuscript. This work was supported by grant GM19670 from the National Institutes of Health. One of us (L. E. M.) was supported in part by the National Institutes of Health training grant, GM07215. The other author (W. S. R.) was supported in part by career development award GM30970 from the Nat)ional Institutes of Health and was a recipient of the Harry and Evelyn Strenbock Career Development Award.

Department of Biochemist,ry College of Agricultural and Life Sciences University of Wisconsin-Madison Madison, Wise. 53706, U.S.A.

LYNNE E. MAQUATt WII,LIAM S. REZNIKOFF

Rcbceivcd 19 November 1979, and in revised form 22 January 1980

REFERENCES

Dickson, R. D., Abelson, J., Barnes, W. M. & Reznikoff, W. S. (1975). Science, 187, 27-35. Gilbert,, W. (1976). In RNA Polymeruse (Losick, R. & Chamberlin, M., eds), pp. 193-205,

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Maizels, N. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3583-3589. Majors, J. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 4394-4398. Maquat, L. E. & Reznikoff, W. S. (1978). J. Mol. Biol. 125, 467-490. Maquat, L. E.. Thornton. K. & Reznikoff, W. S. (1980). J. Mol. Biol. 139, 537-549. Pribnow, D. (1975). J. Mol. BioZ. 99, 419-443. Reznikoff, W. S. (1976). In RNA Polymeruse (Losick, R. & Chamberlin, M., eds), pp.

441-445, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Reznikoff, W. S. & Abelson, J. (1978). In The Operon (Miller, J. H. & Reznikoff, W. S.,

eds), pp. 221-243, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

t Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wise. 53706, U.S.A.

556 L. E. MAQUAT AND W. 8. REZNIKOFF

Rosenberg, M. & Court, D. (1979). Annu. Rev. Genet. 13, 319-353. Schaller, H., Gray, C. & Herrmann, K. (1975). Proc. Nut. Acud. Sci., U.S.A. 72, 737-741. Scherer, G. E. F., Walkinshaw, M. D. & Am&t, S. (1978). Nucl. Acids Res. 5, 3759-3773. Seeburg, P. H., Niisslein, C. & Schaller, H. (1977). Eur. J. Biochem. 74, 1075113.