Proc. Nati. Acad. Sci. USA Vol. 77, No. 12, pp. 7024-7028, December 1980 Biochemistry Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence (promoter/eukaryote/surrogate genetics/synthetic oligodeoxyribonucleotide/fd phage) B. WASYLYK*, R. DERBYSHIREt, A. Guyt, D. MOLKOt, A. ROGETt, R. TEOULEt, AND P. CHAMBON*t *Laboratoire de Cenetique Moleculaire des Eucaryotes du Centre National de la Recherche Scientifique, Unit6 184 de Biologie Mol6culaire et de G6nie Gen6tique de l'Institut National de la Sante et de la Recherche M6dicale, Institut de Chimie Biologique, Facult6 de M6decine, Strasbourg 67085 CUdex, France; and tDepartement de Recherche Fondamentale, Laboratoire de Radiochimie, Centre d'Atudes Nucl6aire de Grenoble-85X, 38041 Grenoble CUdex, France Communicated by E. Peter Geiduschek, August 12,1980 ABSTRACT A single-point mutation, consisting of a T-to-G box is implicated in the promotion of transcription, we have transversion, was made in the third nucleotide of the conalbu- used in vitro site-directed mutagenesis to make a single-base- min gene T-A-T-A-A-A-A homology sequence (the T-A-T-A or Goldberg-Hogness box). In an in vitro system, specific tran- pair substitution in the T-A-T-A box. In this paper we report scription of the mutant DNA was drastically decreased com- the effect on specific in vitro transcription of converting the pared to the normal gene. This down-mutation is consistent with second T of the conalbumin T-A-T-A box to a G. the idea that the T-A-T-A box is an important element for spe- cific initiation of transcription. MATERIALS AND METHODS Synthesis of the Undecadeoxyribonucleotide d-CT-T-T- Recently, specific initiation of transcription of eukaryotic genes T-CT-A-G-A-G. The general materials and methods used for has been obtained in vitro with RNA polymerase B (or II). the preparation and purification of the undecadeoxyribonu- Initially, Weil et al. (1) found that the cell-free system (S100 cleotide were as described (ref. 9 and refs. cited therein). The extract) of Wu (2) conferred specificity on the transcription of sequence of the purified undecanucleotide was determined by the adenovirus 2 major late transcription unit DNA. With the the method of Maxam and Gilbert (10). The 5'-terminal nu- same S100 extract, specific in vitro initiation of transcription cleotide was identified by analysis of the mixture obtained by on the chicken conalbumin and ovalbumin genes has been complete digestion of the 5'-32P-labeled product with snake demonstrated (3). With this system, and in vitro genetic tech- venom phosphodiesterase (9). The sequence of the undecanu- niques to construct deletion mutants, it has been shown (4) that cleotide was also verified by two-dimensional mobility shift sequences, in both the conalbumin gene and adenovirus 2 major analysis (11) of the 5'-labeled product. late transcription unit, located upstream from the mRNA Isolation of Single-Stranded (ss) Bacteriophage fdlO3 DNA startpoints are essential for the initiation of specific transcrip- Containing the Noncoding Strand of the Conalbumin tion. These sequences are located between -44 [i.e., 44 nucle- Pst5-Pst6 Fragment. The conalbumin Pst5-Pst6 fragment (5) otides upstream from the start point for the mRNA (+1), see was cloned into the unique Pst I site of the bacteriophage fdlOS Fig. 1] and -10 for the conalbumin gene and between -32 and vector essentially as described by Herrmann et al. (12) except -12 for the Ad2 major late transcription unit (3, 4). These two that Escherichia colf K514 was used for transfection. Phage regions contain in common a sequence of 12 base pairs, 5' C- plaques were screened for DNA inserts by plaque hybridization T-A-T-A-A-A-A-G-G-G-G 3' [the C is at position -32 (see ref. (12). The orientation of the conalbumin fragments was deter- 5)], encompassing the sequence T-A-T-A-A-A-A (the so-called mined by both restriction enzyme mapping of the replicative T-A-T-A or Goldberg-Hogness box) which is found around form (RF) DNA and by hybridization of the ss-DNA to poly- position -30 in most eukaryotic genes coding for mRNA and (A)+mRNA followed by electron microscopy (not shown). which has been postulated to be a eukaryotic promoter se- Phage was purified from the culture supernatant (prepared by quence (6-8). centrifugation for 10 min at 12,000 X g). After precipitation Supporting this possible role is the observation that, although with 3% polyethylene glycol in the presence of 0.5 M NaCl (12), the adenovirus 2 deletion mutant lacking the sequences up- the phage pellet was redissolved in 50 mM Tris-HCl, pH 7.9/1 stream from position -32 of the gene is fully active in directing mM EDTA (10 ml/500 ml of original culture volume) and specific initiation of in vitro transcription, a mutant with a layered onto gradients constructed with 4.8 ml of 38% (wt/wt) deletion to position -29 is inactivate. This result, however, must CsCl, 4.8 ml of 30.5% CsCl, 4.8 ml of 27% CsCl, and 9.6 ml of be interpreted with some caution. In deletion mutants the DNA 12% (wt/vol) sucrose (all dissolved in 50 mM Tris-HCl, pH upstream from the deletion end point is different from the 7.9/1 mM Na EDTA) in a Beckman SW27 polyallomer tube original DNA, making it impossible to rule out that the observed (one gradient per 500 ml of original culture volume). After effects are due to some inhibitory effect of the replacing se- centrifugation for 16 hr at 130,000 X g and 100C, the phage quences rather than to an alteration of the promoter. band was isolated and dialyzed against 50 mM Tris-HCl, pH In order to obtain unequivocal evidence that the T-A-T-A 7.9/1 mM EDTA. ss-DNA was prepared by phenol extraction followed by ethanol precipitation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: ss, single-stranded; ds, double-stranded; RF, replicative vertisement" in accordance with 18 U. S. C. §1734 solely to indicate form. this fact. t To whom reprint requests should be addressed. 7024 Downloaded by guest on October 2, 2021 Biochemistry: Wasylyk et al. Proc. Natl. Acad. Sci. USA 77 (1980) 7025 -31 -25 RESULTS 1 Pst5 EktNI TATAAAA Heel Pst 6 Construction of Mutant Containing Single-Base-Pair \ Substitution in the Conalbumin T-A-T-A Box. It has been I~~~~ I shown (13-17) that a specific point mutation can be obtained -270 -44 +1 +160 +203 with a synthetic oligonucleotide differing in one base from a FIG. 1. Restriction enzyme map of the conalbumin Pst5-Pst6 wild-type DNA sequence. This oligonucleotide is first hy- fragment. Pst5 and Pst6 refer to Pst I restriction sites as defined (5). bridized to the complementary wild-type strand contained in DNA sequences in the direction of transcription (downstream) are a circular ss-DNA vector, extended with DNA polymerase I, numbered with positive integers; sequences 5' to the start point and finally ligated with DNA ligase to complete the other (upstream) are given negative values. +1 represents the position of strand. Transfection with these molecules gives rise to clones the base coding for the first nucleotide of conalbumin mRNA. T-A- DNA desired sequence We have T-A-A-A-A is the sequence of the noncoding DNA strand between containing with the change. nucleotides -31 and -25 and is the conalbumin sequence equivalent used a fdlOS phage recombinant containing the conalbumin to the T-A-T-A box consensus sequence found in many eukaryotic Pst5-Pst6 fragment (Fig. 1) and a synthetic undecanucleotide genes transcribed by RNA polymerase B (7). to construct a mutant containing a single base substitution in the conalbumin T-A-T-A box (Fig. 2). The noncoding strand of the conalbumin Pst5-Pst6 fragment Biohazards associated with the experiments described in this (Fig. 1) was cloned in fdlOS ss-DNA at the position of the publication were examined beforehand by the French National unique Pst I site of the RF DNA. The undecanucleotide, Committee; the experiments were carried out under L2-B1 complementary to the T-A-T-A box region except for a C conditions. (boxed nucleotide in the upper left circle in Fig. 2), was syn- -30 Q3- TCT AAAAAG C AGA T'C1.- TTTGG 3. T GG c G 20 :G T. (Pot5) ON [PsT5-PsT6J I DNA POLI (Pat55 3.(Pste) I FD 103 ss DNA (KLENOW) T4 DNA LIGASE _ '\ \ G / (4 DNTP, ATP) ,~- 'I~~~~~~'/~~~~~~~~~5 A IG'A AA C\-AT-jTTTCG 3' CTCTG A:T AAA CA XBAISITE C A, _ G CGACC.A.O r UTANT (Put5)) , CON [PsT5-PsT6] (Pst6) (Pat5) pCON [PST5-PST6] FD 103 RF DNIA I \ fD 103 Rf DNA \X \Pstsj-j- [Cowi I I '4 / '4 , v / \ s4% , /, - _- FIG. 2. Preparation of a mutant containing a single-base-pair substitution in the conalbumin T-A-T-A box. Undecanucleotide primer (100 pmol) was 5'-phosphorylated in a reaction mixture (50 Al) containing 50 mM Tris-HCl (pH 9.5), 10 mM MgCl2, 5 mM dithiothreitol, 5% glycerol, 500 MCi (1 Ci = 3.7 X 1010 becquerels) of [y-32P]ATP (New England Nuclear) and 5 units of T4 polynucleotide kinase (Boehringer). After 1 hr at 370C, additional T4 kinase (5 units) and ATP (to 100 ,uM) were added and incubation was continued for 30 min at 370C.