Goldfarb 1982

J. Mol. Biol. (1982) 160, 579-591

Transcription in Vitro of an Isolated Fragment of Bacteriophage T4 Genome

ALEXANDER GOLDFARBt

Max-Planck-Institut ftir Biochemie, D-8033 Martinsried bei Miinchen, F.R.G.

JOEL BROIDA ANDJOHNABELSON

Department of Chemistry, University of California at San Diego La Jolla, Cal<f. U.S.A.

(Received 4 March 1982, and in. revised form 18 May 1982)

A 5 x lo3 base-pair EcoRI fragment of bacteriophage T4 DNA carrying genes 1,3, 57, ipZ and subcluster I of the T4 transfer RNA genes was used as template for purified Escherichia c&i RNA polymerase. RNA transcripts were fractionated by gel electrophoresis and identified by priming with specific dinucleotides and by using as transcription template the 5 x lo3 base-pair fragment after cleavage by different restriction enzymes. We detected and mapped four promoters and one terminator, which were expected to be found on this fragment. In addition, two new termination sites were mapped in the leader region between the tRNA genes and their promoters. The transcription signals on the 5 x lo3 base-pair fragment represent different modes of gene regulation in T4. The in witro system that we describe allows direct quantitative analysis of their functioning under controlled conditions.

1. Introduction

The genome of bacteriophage T4 consists of about 166 kbf of DNA, which can accommodate about 200 genes (Wood & Revel, 1976). Because large numbers of T4 genes are expressed simultaneously at any time during bacteriophage development (Rabussay & Geiduschek, 1977), investigation of T4 gene expression at the level of individual genes is difficult. Likewise, in vitro, about half of the T4 genome is transcribed by Escherichia coti RNA polymerase (Khesin et al., 1962; Geiduschek et al., 1966) to yield a complex mixture of transcripts, which can be separated into at least 30 RNA bands by gel electrophoresis (Goldfarb et al., 1978; Ruger, 1978; Goldfarb & Daniel, 1981). The problem posed by the complexity of the T4 genome to the study of its expression can be overcome by using simplified systems based on the recently prepared collections of T4 DNA clones (Velten et al., 1976; Mattson

t Author to whom all correspondence should be addressed at the Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York. N.Y. 10032. U.S.A.

1 Abbreviations used: kb, lo3 base-pairs; bp. base-pairs.

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0022-2836/82/280579-13 $03.00/0 0 1982 Academic Press Inc. (London) Ltd.

580 A. GOLDFARB. .J. BROIDA ANI) I. ABELSOS

Sequenced A Sequenced B

(bl PsfI t t

H/ncll -1 t

0 I 2 3 4 5 kb I I I I I

FIG. 1. The 5 kb &oRl fragment of T4 genome. (a) A scheme of the 5 kb fragment vit,h boxes representing the 4 structural genes coding for proteins, genes 1. 3, 57 and ipI (internal protein I), and the first subcluster of the T4 tRNA genes (Fukada et al.. 19806). The two segments whose nucleotide sequence is known are indicated by lines A (Fukada & Abelson, 1980) and B (V&en. 1981). Filled circles represent the 4 promoters, I’,. P,, P, and PL, with arrows pointing in the direction of transcription; t indicates a terminator. The locations of P,, P,, t. gene I and tRNA genes are confirmed by the nucleotide sequence (V&en, 1981; Fukada & Abelson, 1980; Mazzara et 01.. 1981); I’, was mapped as an RNA polymerase binding site (Fukada et (LI., 198Oh). In contrast, the locations of genes 3,5i (V&en. 1981) and ipl (Black, 1974), and of promoter P, (Goldfarb & Daniel. 1981; Goldfarb. 1981rz) arr approximate. (b) A restriction enzyme map of the 5 kb fragment, (Fukada rl al.. 1980n.b). The scale below shows distances in kb.

et al., 1977; Wilson et al., 1977; Velten & Abelson, 1980). In this paper we describe the first in vitro system used to analyze several functional T4 transcription signals present on an isolated fragment of T4 DNA.

The 5 kb EcoRI fragment of T4 DNA studied in this work is shown on the scheme of Figure 1. The molecular cloning of this fragment, the mapping of its restriction sites, and its partial nucleotide sequence have been reported previously (Fukada et al., 198Oa,b; Fukada & Abelson, 1980). We know about t,he following transcription signals in this fragment. Promoters P, and P, were inferred from RNA bands appearing among the in vitro transcripts of the whole T4 DNA. These transcripts were mapped by deletion analysis and, on the basis of their sizes, the distance between the two promoters was estimated to be 300 to 600 base-pairs (Goldfarb & Daniel, 1981; Goldfarb, 1981a). Promoter P, was first detected as an RNA polymerase binding site (Fukada et al., 1980b). The location of promoters P, and P, was established from the sizes of “runoff” transcripts synthesized on short DNA restriction nuclease cleavage fragments. The DNA sequence in the region of P, and PL displays canonical promoter sequences in the expected positions (Velten, 1981; Velten & Abelson, unpublished data). A weak terminator, t, was inferred from RNA bands made on whole T4 DNA (Goldfarb & Daniel, 1981). Subsequent nucleotide sequence analysis showed a weak terminator sequence in the expected area (Mazzara et al., 1981).

The analysis of organization and in vitro expression of the right-hand part of the 5 kb fragment (beyond 25 kb on the map of Fig. 1) will be published elsewhere (Velten & Abelson, unpublished data). In this work, our interest was directed mostly to the left half of the fragment, between 0 and 25 kb on our map. This region carries seven tightly linked transfer RNA genes (T4 tRNA gene subcluster I) and gene ipl (internal protein I). The exact position of the tRNA genes follows from the nucleotide sequence (Fukada & Abelson, 1980; Fukada et al., 198Ob), while

TRANSCRIPTION OF T4 RESTRICTION FRAGMENT 581

the position of gene ipI is known only approximately from genetic mapping (Black, 1974).

2. Materials and Methods

(a) Chemicals and enzymes

Ribonucleoside triphosphates and dinucleoside 3’5.monophosphates were from Sigma. [w~‘P]CTP was from New England Nuclear. RNA polymerase isolated from E. coli according to Zillig et al. (1970) was a gift from Barry Chelm. Restriction endonucleases were from New England Biolabs.

(b) DXA templates

The isolation and properties of @tT4 hybrid phage carrying the EcoRI 5160 bp fragment (the 5 kb fragment) are described elsewhere (Fukada et al., 198Ob). The phage was grown and the 5 kb fragment was isolated and purified as described (Fukada et al., 198Ou,b).

The 5 kb fragment was cleaved by the restriction enzymes under the optimal conditions recommended by the manufacturer in lo-p1 reaction mixtures containing 05 pg of DNA and O-5 units (15’ units) of a restriction enzyme. After I h of incubation, the same amount of the enzyme was added and the samples were incubated for another hour. The samples were then heated at 65°C for 10 min and the ingredients of the transcription mixture were added to the same tubes.

(c) Transcription

The standard 100 ~1 transcription system contained 05 pg of DNA, 64 mM each of ATP, GTP, UTP and [32P]CTP (602 Ci/mmol), 40 mw-l’ris.HCl (pH 7-9) 10 mM-MgCl,, 250 ITlM- KCI, 7 mM-2-mercaptoethanol and 1 pg of E. co& RNA polymerase. The reaction was started by adding RNA polymerase to samples prewarmed to 37°C. After 3 min of incubation, rifampicin at 30 pg/ml was sdded and incubation was continued for 20 min.

In the dinucleotide initiation experiments, RNA polymerase was incubated for 5 min at 37°C in the standard mixture without the 4 NTPs but with the indicated priming dinucleotide at 025 mM. ATP, UTP, GTP and [32P]CTP (2 Ci/mmol) were then added at 5 PM each. After 3 min, rifampicin at 30 pg/ml was added, and incubation was continued for 15 min. ATP. UTP, GTP and CTP were then added to 0.4 mM each, and the incubation was continued for another 10 min. The reactions were stopped and the samples prepared for gel electrophoresis as previously (Goldfarb & Daniel, 1981) but the treatment with DNAase was omitted.

(d) Product analysis

The transcripts were analyzed by electrophoresis in non-denaturing composite polyacrylamide/agarose (2.25%/0.5%) g e s as described by Goldfarb & Daniel (1981) or in 1 denaturing polyacrylamide gels containing 8 M-urea as described by Barkay & Goldfarb (1982) except that 3.5% polyacrylamide was used instead of 4%. The denaturing 10% polyacrylamide gel (Goldfarb t Daniel, 1981) contained 6.6 M-urea. The gel slabs were autoradiographed at -70°C with Cronex (DuPont) intensifying screens,

3. Results

(a) In vitro transcripts of the 5 kb DNA fragment

When the 5 kb EcoRI fragment of T4 DNA (see Fig. 1) was transcribed by E. coli RNA polymerase, five high molecular weight transcripts were detected by gel electrophoresis (Fig. 2, lanes 1 and 2). From their approximate sizes, as judged by their mobility relative to ribosomal RNA markers, one can make preliminary

19

582

23 S

A. GOLDFARB. J. BROIDA AND <J. ABELSON

FTC:. 2. Autoradiograms showing the in vilro transcripts of the 5 kb fragment either intact (lanes 1 and 2) or cleaved by different restriction enzymes (lanes 3 to 5). Separation in a 3$i’& polyacrylamide,/urea gel (lane 1) or in a composite polyacrylamide/agarose gel (lanes 2 to ,5). The positions of 23 S and 16 S rRNAs of E. roli in the 3..5% gel are indicated.

assignments of these transcripts to the known control sites on the template DNA fragment. Bands I (~3.5 kb), II (~2.5 kb) and III (~2 kb) have sizes that might be expected of the runoff transcripts initiated at the leftward promoters P,, P, and P,, respectively. Band IV ( g l-6 kb) is likely to result from occasional termination at the site t of the chains initiated at P,, which is the strongest of the leftward promoters. The two other bands, which can be expected to arise from the t- mediated termination of the leftward transcription initiated at P, and P,, are not seen, presumably because they should be relatively weak or masked by other bands. Band V (g 1.2 kb) is likely to be the runoff transcript initiated at the rightward promoter P,.

This assignment of bands I to V was confirmed by the pattern of transcripts


obtained when the template DNA fragment was cleaved with different restriction enzymes prior to transcription (Fig. 2, lanes 3 to 5). As can be expected from the restriction map (Fig. l(b)), cleavage of the template with all three restriction enzymes tested results in the disappearance of band I from the products of transcription ; transcripts II and III are affected by template cleavage with H&II and AvaII and bands IV and V are not made on the template cleaved with AwuII. The cleavage with HincII and AvuII results in the formation of characteristic pairs of new transcripts, which are, apparently, the new shorter runoff products initiated at promoters P, and P,.

(b) Dinucleotide priming of the transcripts

It is known that RNA polymerase can selectively initiate transcription at a given promoter if it is provided with a dinucleotide primer complementary to the DNA sequence around the RNA starting site, while the concentration of the four standard nucleoside triphosphates is kept low (Minkley & Pribnow, 1973). It was shown previously that the T4 promoter P, responds to selective initiation with dinucleotides UpA and ApU. Promoter P, can utilize primers CpA and ApC, which presumably reflects the sequences T-A-T and C-A-C, respectively, at their initiation sites (Goldfarb, 1981a). The nucleotide sequences of promoters P, and P, have been determined (Velten, 1981; Velten & Abelson, unpublished data). In the areas relevant to our discussion they are as follows:

T-A-T-A-A-T-C-T-T-A-A-T-T-A-i-A-T-T-T... (Pm) and

. T-A-T-T-A-T-C-T-A-A-G-A-T-G-T-A-A-A-G... PL).

in which “Pribnow boxes” are underlined and initiation starts (established by J. Velten) are designated by arrows. From these data one can deduce which nucleotides should be good primers for promoters P, and P,. Thus in our next experiment we primed the transcripts described in the preceding section with different dinucleotides (Fig. 3).

The results demonstrate that the transcripts of the 5 kb fragment (with the exception of band I, see below) respond to the dinucleotide priming in accordance with their preliminary assignment to the promoters in the preceding section. Band III can be initiated with UpA and ApU, the two primers specific for the promoter P,. Band IV, which is presumably initiated at the same promoter, responded well to initiation with UpA. At longer autoradiogram exposure (data not shown), the priming of band IV with ApU was also detected. Band II was initiated with CpA, the primer specific for the promoter P,. Another high molecular weight band (band VI), which is slightly shorter than band III, was initiated with CpA. This band apparently results from termination at the site t of chains initiated at P,. Band V responded to priming with ApU, UpG and GpU, which may reflect the sequence A-T-G-T around the starting site of its promoter. Indeed, such a sequence is present in position 6 to 9 downstream from the Pribnow box of the promoter P, (see above). Several other dinucleotides including ApA, which should presumably

584 A. GOLDFARB, J.

123456

BROlDA AND J. ABELSON

m s

UpA ApU CpA UpG GpU ‘0 E iJi

FIG. 3. Dinucleotide initiation of RNA chains on the 5 kb fragment. RP;A was synthesized either under standard conditions (lanes 1 and 7) or under the conditions of selective chain initiation with the indicated dinucleotides (lanes 2 to 6 and 8 to 12). Separation was in a 3.5% denaturing gel (lanes 1 to 6) and in a composite gel (lanes 7 to 12). The positions of E. coli rRNA markers in the 3.5% gel are shown. Roman numbers designate transcript bands discussed in the text.

prime band I, were tested and none of them stimulated initiation of RNA chains from the 5 kb fragment (data not shown). Our failure to detect the dinucleotide priming of band I is probably due to the general weakness of its promoter (note the low intensity of band I relative to other transcripts in Figs 2 and 3).

In addition to bands I to VI, several new transcripts were detected when RNA synthesis was performed under the dinucleotide priming conditions (bands VII, VIII and IX in Fig. 3). The appearance of a new band can, in principle, result either from a new initiation or a new termination event. The response of band VIII to priming with UpA and ApU suggests that it may be initiated at promoter P, and, consequently, may represent premature termination of the leftward

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transcription. This suggestion is supported by the gel position of the new band VII : if this band is initiated at promoter P,, as suggested by its response to CpA priming, it should be terminated at the same site as band VIII, since the size difference bettieen bands VII and VIII is about the same as the distance between promoters P, and PZ.

(c) TranscriptioTa of the 5 kb fragment cleaved with restriction enzymes

To map the UpA and CPA-primed transcripts more precisely, the dinucleotide initiation experiment was performed using as template the 5 kb fragment, which was cleaved with different restriction enzymes. The results are presented in Figure 4 and Figure 5, for UpA and CPA, respectively.

The pattern of UpA-initiated transcripts made on a series of restriction enzyme digests of the 5 kb fragment (Fig. 4(a)) can be explained only by the scheme in Figure 4(b), whereby bands III, IV and VIII and the series of the runoff transcripts (the slowest-moving bands in each lane) are all initiated at the same site (Pi). Thus bands IV and VIII apparently result from premature termination of the leftward transcription. Similarly, the scheme in Figure 5(b) is the only possible explanation for the transcripts made on the cleaved 5 kb fragment under the conditions of CpA initiation. Transcripts II, VI, VII and IX, as well as the runoff products, are all initiated at the same site (P2), and bands VI, VII and IX apparently result from termination of the leftward transcription at sites between the promoter P2 and the end of the restriction fragments. The data of Figures 4 and 5 map the two promoters and the termination sites relative to the restriction sites on the 5 kb fragment. Clearly, bands IV and VI, as well as bands VII and VIII, result from the termination of RNA chains at the same two sites. The approximate location of the promoters Pi and P, from these data are about 2.0 kb and 2.4 kb, respectively.

To map the two promoters more precisely, we performed the experiment shown in Figure 6. The 5 kb fragment cleaved with restriction enzymes Tag1 or AEuI was transcribed using UpA or CpA, respectively, as primer, and the products were analyzed on a 10% polyacrylamide/urea gel, which allows one to visualize low molecular weight RNAs. The runoff transcripts produced in these two reactions had lengths of about 40 and 70 nucleotides, respectively, as judged by their migration relative to stable RNAs or E. coli. This result locates the starting point of the promoter P, about 40 nucleotides from the end of the 400-bp TaqI fragment, and t’he starting point of P, about 70 nucleotides from the end of the 340-bp AZuI fragment (Fig. 6(b)). Therefore, the distance between the two promoters is about’ 330 bp. Our result is in excellent agreement with the data of Fukada et aE. (l%Ob), who found an RNA polymerase binding site within the 100-bp AluI fragment shown in Figure 6(b).

4. Discussion

(a) Transcription sites on the 5 kb fragment

The results of our experiment demonstrate directly the functioning of several transcription sites on the 5 kb fragment. Although we did not make a detailed

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FIG. 6. Fine mapping of promoters Pi and P,. (a) Autoradiogram showing the transcripts synthesized on the 5 kb fragment that was cleaved with restriction enzymes Z’aqI and AIuI under the conditions of selective chain initiation with UpA and CpA, respectively. Separation is in a loo/, denaturing gel. The right-hand lane contains separated stable RNAs of E. co&. (b) A scheme showing a part of the 5 kb fragment between positions 1.9 kb and 25 kb. The restriction maps for AM and TqI are shown (Fukada et al., 19ElOu,b) with the fragment lengths in bp. Vertical broken lines indicate the positions of the starting points of promoters P, and P2, and their projections on the restrict,ion map. The 2 runoff transcripts seen on the autoradiogram in (a) are represented by arrows.


comparison between their activities on the isolated restriction fragment and on the whole T4 DNA, we note that the efficiency of initiation at promoters P, and PZ and the frequency of termination at the weak terminator t are about the same in our present experiments and in those on the intact T4 DNA template (Goldfarb & Daniel, 1981; Goldfarb, 1981a). We conclude, at least with regard to these three sites, that it is of little importance in vitro whether or not the template contains glucosylated hydroxymethylcytosine, which is a normal component of T4 DNA (Wyatt & Cohen, 1952; Lehman & Pratt, 1960).

Our results map several transcription signals relative to the restriction map. In the direction from left to right on our map we find a terminator (t) at 0.4 kb, which is responsible for the formation of bands VI and IV; at about 1.6 kb, there is a terminator of bands VII and VIII ; and at about 20 kb there is (are) a terminator(s) of multiple band IX. The two leftward promoters, Pi and PZ, are mapped around 2.0 and 2.35 kb, respectively (Fig. 6(b)). The positioning of the transcription sites relative to the restriction enzyme map will be an important aid in the choice of fragments for subcloning, sequencing and setting up of in vitro systems. For example, the runoff transcripts synthesized on the restriction fragments carrying promoter P, were used recently for the mapping of RNAase III processing sites in this area (Barkay & Goldfarb, 1982).

Our mapping data pose several interesting questions about the arrangement of functional sites in the P, to P, region: (1) what is the relation between the promoter P, and the multiple terminator that we map at about 2.0 kb? (2) Is the 2.0-kb terminator related to an RNAase III cleavage site, which was mapped about 40 nucleotides downstream from Pi and should involve a region of dyad symmetry in the sequence (Barkay & Goldfarb, 1982) ‘z (3) What is the position of the coding sequence of ipI (about 240 bp for this 80 amino acid protein; Isobe et al., 1977) relative to P,, Pi, the 29kb and the 1.6-kb terminators, and the RNAase III sites downstream from P, ? The answers to these questions will come from the nucleotide sequence of this region, which is expected to emerge soon from the current efforts of several groups.

(b) Conditional termination at 1% and 2-O kb

The transcription termination sites that cause the formation of bands VII and VIII (at 1.6 kb), and the multiple band IX (at 2-O kb), deserve special discussion. These bands appear only if transcription is performed under dinucleotide initiation conditions and are not seen among the products of the standard transcription system (Fig. 3).

One possible suggestion is that, under the selective priming conditions, the termination at 1.6 and 2-O kb results from the “bumping” of the elongating transcription complex into another RNA polymerase molecule bound to a promoter but unable to initiate due to the absence of the appropriate primer (Axelrod, 1976; Kassavetis et al., 1978). This scheme is favored by the fact that the 2.0-kb terminator and promoter P, map very close to each other. One has to assume a new RNA polymerase binding site to account for the l-6-kb terminator, according to this scheme. The bumping model, however, is not supported by the result of our

590 A. G0LDFBRB, J. BROIDA AND J. ABELSON

preliminary experiment designed to test it with regard to the 2.0 kb site: lowering of the RNA polymerase/template ratio in the CpA initiation protocol did not abolish the appearance of the multiple band IX (data not shown).

Alternatively, one can suggest that termination at the 1.6 and 2.0 kb sites is triggered by the pausing of RNA polymerase (see Adhya & Gottesman. 1978), caused by the low nucleotide concentration that is employed at the first step of the dinucleotide initiation protocol. This phenomenon, however, cannot be reduced to simple pausing, since RNA chain elongation is not resumed efficiently upon subsequent incubation with high nucleotide concentrations. The termination sites at 1.6 and 2.0 kb can probably direct chain release but) require low nucleotide concentration for the initial pausing of RNA polymerase. At present. we do not, have any hints on the possible biological role of the terminators at 1.6 and 2.0 kb.

(c) The 5 kb fragment as a?, experimental system

The 5 kb EcoRI fragment, whose i,n vitro transcription products we have described, is of considerable interest in the study of T4 infection mechanisms, because its genes represent all major types of T4 transcription control classes (Rabussay & Geiduschek, 1977). The first tRNA subcluster and ipI were classified as immediate-early genes (Black, 1974; Goldfarb & Daniel. 1980; Young et al.. 1980). The terminator t is thought to be the boundary between immediate and delayed-early transcription (Goldfarb &Z Daniel. 1980,198l). Promoter P, is not utilized by T4-modified RNA polymerase while promoter P, is (Goldfarb, 1981b: Goldfarb & Palm, 1981). Gene I specifies a middle function and gene 3 a late function (Cohen et al., 1974; Wood & Revel, 1976: Mattson et al.. 1978; Linder Nr Skold, 1980). That genes”1 and 3, whose expression in viva needs T4specific factors, are transcribed at all by the host polymerase (see also Natale &, Buchanan, 1977) seems quite surprising, and this will be discussed in depth else ere (Velten Hr Abelson, unpublished data).

In conclusion. our results define an in vitro system that will allow direct quantitative analysis of transcription control in this interesting region.

We thank Barry Chelm for a gift of RNA polymerase and Dave Figurski for his comments on the manuscript. A.G. is grateful to Professor WI. Zillig for support and discussion and to the members of J. Abelson’s group for their hospitality during his stay in La *Jolla. This work was partially supported by National Institutes of Health grant CA10984. A.(:. is a recipient of a Max-Planck Fellowship.

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Goldfarb 1982