In vitro synthesis of T4 proteins: Control of transcription of gene 57

VIROLOGY

43, 209-213 (1971)

In Vitro

Synthesis

of T4 Proteins: of Gene

JAMES Department

M. WILHEL?\/I’ of Biophysics,

AND

University

Control

of Transcription

57 ROBERT

HASELKORN

of Chicago, Chicago, Illinois

60637

Accepted September 28, 1970 in RNA prepared from Escherichia coli In vitro suppression of an amber mutation infected with a mutant in T4 gene 57 proves that a particular band observed on acrylamide gels is the product of gene 57 (P57). The capacity of RNA preparations to direct the synthesis of P57 in vitro has been used to study the regulation of transcription of gene 57. Gene 57 message is absent from RNA made on a T4 DNA template in vitro, and from RNA prepared from cells infected with T4 in the presence of chloramphenicol. It is present in early RNA from cells infected with wild-type T4, a DNA-negative mutant, and a maturation-defective mutant. It is present more abundantly in late RNA in these three infections. These results suggest that gene 57 message is a member of the class termed “postreplicative early” RNA. INTRODUCTION

Detailed DNA-RNA hybridization-competition analyses permit a considerable extension of the simple ‘Learly”-“late” distinction in the program of gene expression during bacteriophage T4 infection (Salser et al., 1970). One class of messenger RNA detected in t,he newer analyses (originally termed “quasi-late” but now called “postreplicative early”) is synthesized early (prior to 5 min at 30”) and increases in abundance after viral DNA replication, in contrast to the true early RNA whose synthesis is turned down after DNA replication begins. While the postreplicative early RNA appears to be a quantitatively significant fraction of the RNA present at 5 min, very few proteins have been specifically tletected with a corresponding pattern of regulation. One possible candidate is the product of gene 57, which we shall call P57. In an extensive analysis of protein synthesis in T4 infection by polyacrylamide gel electrophoresis, Hosoda and Levinthal (1968) found that P57 is synthesized throughout infection. 1 Present address: Department biology, University of Pennsylvania, phia, Pennsylvania.

of

MicroPhiladel209

We have previously shown that I’57 may be synthesized in an in vitro system directed by RNA from T4-infected E. coli (Wilhelm and Haselkorn, 1969). We report here the proof, by in vitro suppression of an amber mutant in gene 57, that the band on acrylamide gels previously identified as P57 is in fact the primary protein product of that gene. Based on in vitro synthesis of P57, we find that the gene 57 message follows a transcription program different from both the true early and true late RNAs defined by DNA-RNA hybridization-competition analyses. Gene 57 message appears to be a member of the class termed postreplicative early. MATERIALS

AND

METHODS

Bacteria and bacteriophages. E. coli BE was the nonpermissive host and CR63(Su1+), the permissive. The amber mutants of T4 have been described (Epstein et al., 1963) and are listed below: gene 45, am ElO; gene 55, am BL292; gene 57, am E198. Protein synthesis. Our procedures for the preparation of radioactive lysates, the preparation of cell-free extracts, and the conditions of protein synthesis in. vitro are described in the preceding paper (Wil-

210

WILHELM

AND

helm and Haselkorn, 1971). Leucine-14C was the only amino acid used to label proteins, except in the experiments of Fig. 1, for which the in vitro incubation contained a mixture of ten 14C-amino acids. Also, the protein synthesis system was prepared according to Capecchi (1966), and in vitro synthesis was carried out as described by Wilhelm and Haselkorn (1969). Analysis of proteins by electrophoresis on polyacrylamide gels. The standard discontinuous gel system of Hosoda and Levinthal (1968) was employed. For analysis of the in vitro products, a postribosomal supernatant was obtained by centrifugation of the in vitro system at 37,000 rpm for 90 min in the Spinco No. 40 rotor. Of the total radioactivity incorporated into protein, usually 30-40% remains in the supernatant after centrifugation of the ribosomes. Proteins were fixed in the gels after electrophoresis by immersing them in a solution of 1% Naphthol Blue-Black in 7.5 % acetic acid; the stain was removed by several changes of acetic acid. Note that both the gel system (no SDS) and the centrifugation after in vitro synthesis differ from the procedures used in the preceding paper. These differences were necessary because we have been unable to locate P57 on SDS gels to date. RESULTS

RNA isolated late in infection with wildtype T4 directs the synthesis, in vitro, of a protein whose mobility on standard acrylamide gels corresponds to that of P57 (Wilhelm and Haselkorn, 1969). The pattern of proteins directed by wild-type T4 RNA is compared with the pattern of proteins directed by RNA isolat,ed from cells infected with an amber mutant in gene 57 in Fig. 1. When in vitro protein synthesis directed by the mutant RNA is carried out in cell-free extracts prepared from nonpermissive cells, the band corresponding to P57 is missing (Fig. 1, d and e). When in vitro protein synthesis is carried out in extracts from permissive cells, the in reduced band is present, although amounts (Fig. 1, b and c). These results prove that the band in question is P57; com-

HASELKORN

Mlgratlon

Distance

(cm)

FIG. 1. Patterns of proteins synthesized in vitro with 19 min RNA from Escherichia coli infected with wild-type T4 or an amber mutant in gene 57. The data are shown as microdensitometer tracings of the autoradiograms. For each tracing, the following conditions are specified: the type of RNA (type of cell infected); type of cell used for preparation of the protein synthesis system; the amount of radioactivity in the prot,eins applied to the gel. (a) wild type (Su-); SIC; 7260 cpm. (b) 57- (Su+); Su+; 3120 cpm. (c) 57- (Su-); Su’; 2760 cpm. (d) 57- (Su+); SC, 7400 cpm. (e) 57(Su-); Su-; 5100 cpm. 0.4 ml systems were programmed with 150-230 pg RNA in each case. At this level, RNA is still limiting.

parison of gels b and c suggests, moreover, that the abundance of gene 57 message is similar in permissive and nonpermissive infection with am E198. Therefore degradation of gene 57 message distal to the amber

I,\;

VITRO

SYNTHESIS

OF T4 PROTEINS:

mutation in am El98 is no more rapid in E. coli B than in CR63. All the late RNA preparations direct the synthesis of one other major protein distinguished by the standard gel system. That is the large band in the center of the pattern; we have already demonstrated, by in vitro suppression, that it is the product of gene 22 (Wilhelm and Haselkorn, 1969). The gene 22 product is a true late protein; its message is not detected in early RP\‘A from wild-type infection, or in late RNA from DNA-negative or maturation defective mutant infection. Although the gene 57 protein functions late in infection, it is present early (Hosoda and Levinthal, 1968). The protein plays a regulatory role in the formation of tail fibers (Edgar and Lielausis, 1965), probably at the level of assembly (S. Ward, personal communication). Since we were able to detect the synthesis of P57 in vitro, we studied the control of synthesis of the gene 57 message. Early T4 RNA can be subdivided into two classes on the basis of a requirement for postinfection protein synthesis (Salser et al., 1970). In Fig. 2(a) we show the proteins synthesized in vitro directed by RNA isolated from cells infected with wild-type T4 for 19 min in the presence of chloramphenicol. P57 is absent; on this basis we conclude that P57 is not an “immediate early” protein, but one whose message transcription requires prior viral protein synthesis. Many of the “delayed early” RNA species, which require prior viral protein synthesis in vivo, are transcribed from T4 DNA in vitro by E. coli RNA polymerase (Milanesi et al., 1970). Such RNA preparations, synthesized in vitro in a system coupled with protein synthesis, direct the subsequent synthesis of T4 (Y- and fi-glucosyl transferase and lysozyme (Gold and Schweiger, 1969: Schweiger and Gold, 1969) as well as a number of other T4 early proteins, including some of molecular weight greater than 100,000 (our unpublished observations). Nevertheless, they fail to direct the synthesis of P57 in vitro (Fig. 2, b). Since delayed early RnTA is probably made in vitro by a mechanism that permits the readthrough of normal termination signals (Milanesi et al.,

GENE

57 TRANSCRIPTION

in VI’~O protein

211

synthesis

I (0) CAP- RNA

I P57

--i Migration

distance

(cm)

FIG. 2. Patterns of proteins synthesized in vitro (a) with RNA isolated 19 min after infection with T4 in the presence of chloramphenicol or (b) in an in vitro system programmed with DNA. In (b) the protein synthesis system was prepared without pancreatic deoxyribonuclease. The incubation contained the usual components plus UTP, CTP, and excess GTP each at a concentration of 0.5 mu. The proteins were labled only with leucine-‘4C. The amount of radioactivity in proteins applied to the gel was 2300 cpm. The dried gel was exposed to X-ray film for 16 days. In (a), proteins were labeled with a W-amino acid mixture and 5800 cpm were applied to the gel. Exposure time was 8 days.

1970), this result suggests that either (a) gene 57 is transcribed clockwise, as are the majority of late genes, or (b) it is transcribed counterclockwise from a promoter that is upstream with respect to all true early promoters. RNA isolated 6 min after wild-type T4 infection directs the synthesis of P57, although in smaller amounts than 19 min RNA (compare Fig. 3, a and e). We believe the increase in relative amount of P57 among the products of late RNA directed protein synthesis reflects an increase in abundance of gene 57 message late in infection. This increase in abundance does not require viral DNA replication (Fig. 3, c). Moreover, infection with a mutant in gene 55, in which viral DNA replication is normal but late

212

WILHELM

AND HASELKORN

wild-type T4 infection. True late transcription begins after DNA replication, around 9 min, and requires a functional gene 55 prodt uct (Bolle et al., 1968; Haselkorn et al., 1968). (0) P22 Most of the true late genes are transcribed 19min. am+ RNA 1 II clockwise. Gene 57 message is not detected in chloramphenicol RNA or in RNA made from T4 DNA in vitro; therefore the gene 57 message is neither an immediate early nor a delayed (b)Igmin. MD RNA II II early as defined by Milanesi et al. (1970). Gene 57 message is not a true late RNA either, since it is present at 6 min and does not require the gene 55 product for tran(C) 19 min. DO RNA scription. The increase in abundance late in wild-type infection does not appear to be simply due to gene dosage, since it occurs in both gene 55 infection and in DNA-negative infection (Fig. 3). These results require us to postulate an additional transcription control element in T4 infection. We assume that host RNA polymerase initiates transcription at true I I I I I I I I early promoters and moves counterclock4 2 6 a 0 wise, progressing from immediate early to Migrotion Distance (cm) delayed early genes. The progression from FIG. 3. Patterns of proteins synthesized in vitro immediate to delayed early transcription in with RNA isolated from nonpermissive cells invivo may require modification of termination fected with wild-type T4 and amber mutants in (Milanesi et al., 1970) or of initiation genes 45 and 55. For each tracing the type of (Travers, 1970) or both; the progression in RNA is indicated followed by the amount of radioactivity in proteins applied to the gel. (a) vitro has no such requirements (Milanesi et 19-min wild-type, 1250 cpm; (b) 19 min 55-, 1210 al., 1969). Since P57 is not made from T4 cpm; (c) 19 min 45-, 1430 cpm; (d) 6 min 55-, 1360 DNA in vitro, although many other early cpm; (e) 6 min 45-, 1300 cpm. 0.4 ml systems proteins are, we conclude that gene 57 meswere programmed with 170-250 pg RNA in each sage is not accessible, downstream, from any case. early promoter recognized by host, RNA polymerase in vitro. gene expression is not, also displays nearly Gene 57 is at an interface between early the wild-type increase in abundance of gene (gene 1, mononucleotide kinase) and late 57 message (Fig. 3, b). These results suggest (gene 2, head-filling) genes on the T4 map that the promoter for gene 57, in both (Epstein et al., 1963). If gene 57 is tranparental and replicated DNA, is recognized scribed clockwise, as the true late genes are, by a transcription element different from then there must, be a termination signal for the gene 55 product. transcription between gene 57 and gene 2, to account for the absence of gene 2 (late) DISCUSSION The experiments on in vitro synthesis of message in DO and gene 55 infection. Moreover, the requisite promoter would differ P57 permit us to distinguish the transcripfrom other “clockwise” promoters by its tion of gene 57 from true early genes, on one accessibility on unreplicated, parental DNA. hand, and true late genes, on the other. True Alternatively, if gene 57 is transcribed counearly genes, whether immediate or delayed, terclockwise, its inaccessibility for tranare transcribed only early in infection; their transcription is terminated before 12 min in scription in vitro is understandable, since its in vitro

protein

synthesis

757

IN

VIZ’RO

SYNTHESIS

OF T4 PROTEINS:

location on the T4 chromosome is in fact upstream from the long sequence of early genes beginning at gene 1. DNA-RNA hybridization experiments permit a tentative choice between these alternatives. Postreplicative early RNA hybridizes with both strands of T4 DNA; the increase in abundance late in infection of r-strand (transcribed clockwise) complementary RNA, but not of l-strand complementary RNA, requires viral DNA replication (A. Guha, W. Szybalski, A. Belle, W. Salser, R. Epstein, E. P. Geiduschek, and J. F. Pulitzer, to be published). Since the increase in gene 57 message abundance does not appear to require DNA replication (Fig. 3), we prefer its assignment to the l-strand, or counterclockwise transcription. Direct experiments to test this assignment are feasible. ACKNOWLEDGMENTS This research was supported by a grant from the National Institutes of Health (AI-GM-09315). One of us (J.M.W.) was a postdoctoral fellow of the American Cancer Society. We thank Mrs. A. Tomic for expert technical assistance and Mr. G. Grofman for preparing photographs. REFERENCES BOLLE, A., EPSTEIN, R. H., SALSER, W., and GEIDUSCHEK, E. P. (1968). Transcription during bacteriophage T4 development : Requirements for late messenger synthesis. J. Mol. Biol. 33, 339-362. CAPECCHI, M. (1966). Cell-free protein synthesis programmed with R17 RNA: Identification of two phage proteins. J. Mol. Biol. 21, 173-193. EDGAR, R. S., and LIELAUSIS, I. (1965). Serological studies with mutants of phage T4D defective in gene determining tail fiber structure. Genetics 52, 1187-1200. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E.,

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CHEVALLEY, R., EDQAR, R. S., SUSMAN, M.,. DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. GOLD, L. M., and SCHWEIGER, M. (1969). Synthesis. of phage-specific (Y- and p-glucosyl transferases directed by T-even DNA in vitro. Proc. Nat.. Acad. Sci. U. S. 62, 892-898. HASELKORN, R., BALDI, M. I., and DOSKOCIL, J(1968). RNA synthesis in T4-infected E. coli B. “The Biochemistry of Virus Replication,“’ pp. 79-92. Universitets-Forlaget, Oslo. HOSODA, J., and LEVINTHAL, C. (1968). Protein synthesis by Escherichia coli infected with bacteriophage T4D. Virology 34, 709-727. MILANESI, G., BRODY, E. N., and GEIDUSCHEK, E. P. (1969). Sequence of the in vitro transcription of T4 DNA. Nature (London) 221, 10141016. MILANESI, G., BRODY, E. N., GRAU, O., and GEIDUSCHEK, E. P. (1970). Transcription of the bacteriophage T4 template in vitro: Separation of “Delayed Early” from “Immediate Early” transcription. Proc. Nat. Acad. Sci. U. S. 66, 181-188. SCHWEIGER, M., and GOLD, L. M. (1969). Bacteriophage T4 DNA-dependent in vitro synthesis of lysozyme. Proc. Nat. Ad. Sci. U. S. 63, 1351-1358. SALSER, W., BOLLE, A., and EPSTEIN, R. (1970). Transcription during bacteriophage T4 development : A demonstration that distinct subclasses of the “early” RNA appear at different times and that some are “turned off” at late times. J. Mol. Biol. 49, 271-296. TRAVERS, A. (1970). Positive control of transcription by a bacteriophage sigma factor. Nuture (London) 225, 1009-1012. WILHELM, J. M., and HASELKORN, R. (1969). In vitro synthesis of T4 proteins: Lysozyme and the products of genes 22 and 57. Cold Spring Harbor Symp. Qua&. Biol. 34, 793-798. WILHELM, J. M., and HASELKORN, R. (1971). In vitro synthesis of T4 proteins: The products of genes 9,18,19,23,24, and 38. Virology 43,19&208.