- Email: [email protected]
In vitro synthesis of T4 proteins: The products of genes 9, 18, 19, 23, 24, and 38
VIltOLOGY
43, 198-208 (1971)
In Vitro
Synthesis Genes
JAMES Department
of T4 Proteins:
The Products
9, 18, 19, 23, 24, and
M. WILHELM’ of Biophysics,
ROBERT
AND
38
HASELKORN
of Chicago,
University
of
Chicago,
Illinois
60637
Accepted September 28, 1970 The improved acrylamide gel electrophoresis procedure of Laemmli (1970) permits identification of a number of specific T4 gene products among the proteins whose synthesis is directed in vitro by RNA from T4-infected E. coli. RNA isolated late in infection directs the synthesis of the products of genes 9 (baseplate), 18 (tail sheath), 19 (tail core), 23 and 24 (head), and 38 (tail fiber). Synthesis of these proteins is not directed by RNA isolated early in wild-type T4 infection, or by RNA isolated late in DNA-negative or maturation defective infection. These results are consistent with the suggestion that T4 gene expression is regulated primarily at the level of transcription. A major difference between the products of in vitro protein synthesis and the display of proteins made in viva is the absence of proteins of molecular weight greater than 80,000 from the former. We believe this result is a consequence of transcription-translation coupling in wivo, which limits the size of functional messages that can be isolated. The proteins made in vitro also lack P23*, a protein found in viva as a result of “processing” of the primary product of gene 23. INTRODUCTION
The synthesis of specific proteins in vitro is a useful tool for the analysis of populations of messenger RNA. The technique has been applied most extensively to RNA from Escherichia coli infected with bacteriophage T4, with which the in vitro synthesis of enzymatically active lysozyme (Salser et al., 1967) and the G+ and fl-glucosyl transferases (Young, 1970; Young and Van Houwe, 1970) has been demonstrated. It. is also possible to couple transcription and translation in vitro to direct the synthesis of these activities with T4 DNA (Gold and Schweiger, 1969; Schweiger and Gold, 1969). There are many T4 proteins, especially the structural components of the phage, which have no readily demonstrable enzymatic activities. The synthesis of these gene products in infected cells may be studied by electrophoresis of labeled cell extracts in polyacrylamide gels (Hosoda and i Present address : Department biology, University of Pennsylvania, phia, Pennsylvania.
of
Micro-
Philadel-
Levinthal, 1968). We have used gel electrophoresis to examine t,he products of T4 mRNA-dependent protein synthesis (Coolsma and Haselkorn, 1969) and have reported the specific synthesis of the products of T4 genes 22 and 57 (Wilhelm and Haselkorn, 1969). The gene 22 product is a factor in the control of the shape of the phage head (Laemmli et al., 1970) and gene 57 pIays a regulatory role in the formation of tail fibers (Edgar and Lielausis, 1965), most likely at the level of assembly (S. Ward, personal communication). In the present communication we report the in vitro synthesis
of
a variety
of
other
proteins of T4 directed by RNA from cells after phage infection. MATERIALS
AND
structural
isolated
METHODS
Bacteria and bacteriophages. E. coli BE, the nonpermissive host, was used for preparat’ion of protein-synthesis systems and for all infected-cell RNAs. Stocks of amber phages were grown on E. coli CR63 (Sul+). The amber mutants of T4 have been de-
198
lAr VZTRO
SYNTHESIS
OF T4 PROTEINS:
scribed (Epstein et al., 1963) and are listed here so that they may be referred to later only by gene number: gene 9 defective (9-), arnE17; lo-, B255; 18-, EM; 19-, KG524; 22-, N98; 23-, B17, 24-, E355; 34-, a double mutant, B25-A455; 37-, a double mutant, N52-B280; 3S-, B262; 45-, ElO; 55-, BL292; 57-, E198. Media. For lysates and RNA: M9, which contains, per liter, 7 g Na2HP04. 2Hz0, 3 g KH2P04, 0.5 g NaCI, 1 g NH&I, 1O-3 d1 MgSOd, lop4 M CaC&, and 4 g glucose was used. 119s is the same supplemented with 10 g Casamino acids (Difco). For growth of cells for protein-synthesizing extracts: per liter, 10 g Bactotryptone and 1 g yeast extract (Difco), 8 g NaCl, and 1 g glucose was used. Preparation of radioactive lysafes. E. coli BE were grown to a density of 4 X lo8 cells/ml in Sl9 at 37”, shifted to 30” and supplemented with n-tryptophan (40 pg/ ml). 5 ml of culture were infected at a m.o.i. = 5 and at 20 min after infection the proteins were labeled by addition of 1.25 &i of leucine-14C (315 mCi/mmole). Four minutes later the culture was chilled with ice, the cells collected by centrifugation and the cell pellet was resuspended in 0.5 ml of 0.01 M Tris.HCl, pH 7.6, 0.01 M I\Ig acetate. This suspension was stored frozen. The incorporation of leucine-14C into hot trichloroacetic acid (TCA) -insoluble material was generally 3000-5000 cpm (Beckman Low Beta Gas-flow Counter; efficiency 17%) in a 50-~1 aliquot of culture. Preparation of RI+‘A from infected cells. E. coli BE were grown in M9S to a density of 5 X 10e cells/ml at 30”, supplemented and infected as dewith n-tryptophan, scribed. The RNA was isolated as described by Bolle et al. (1968a) with the following modifications: egg white lysozyme was added to the resuspended cells only when RNA was prepared at 6 minutes after infection or when no endogenous lysozyme is produced (infection with gene 45 or gene 55 mutants) ; the first phenol extraction was performed at 65” for 2 min but the two subsequent extractions were at 37”; after ethanol precipitation, the RNA was resuspended in 0.01 M Tris .HCl, pH 7.6, to an approximate concentration of 5
GENE
PRODUCTS
199
mg/ml and dialyzed against distilled water for 6 hr in the cold. Protein synthesis. Cells were grown in the medium given above at 37” until the turbidity reached 0.7 at 650 nm. The cells were chilled with ice and collected by centrifugation. The cell pellet was washed twice by resuspension in 0.01 M Tris.HCl, pH 7.6, 0.01 M lllg acetate and centrifugation, then frozen and stored overnight. Cell-free extract was prepared and separated into ribosomes and a supernatant protein fraction by the method of Gold and Schweiger (1969) with two modifications: the cells were broken by grinding with alumina (2.5 X the wet weight of cells) and the buffer used for the extraction of the paste of cells and alumina contained pancreatic deoxyribonuclease (Worthington, electrophoretically pure) at 1 pg/ml. In vitro protein synthesis was generally carried out in an incubation volume of 0.4 ml. This contained 1.4 mg ribosomes, 600 pg of supernatant protein fraction, 400 pg of E. coli B transfer RNA (General Biochemicals), 30 pmoles Tris. HCl, pH 7.6, 10 pmoles KCl, 28 pmoles NH&I; 3.4 pmoles MgSO+ 0.5 pmole dithiothreitol, 2 pmoles phosphoenol pyruvate, 5 pg pyruvate kinase, 1.2 pmoles ATP, 0.08 pmole GTP, 6 nmoles each of ‘Y-alanine, -asparagine, -cysteine, -glutamic acid, -glutamine, -histidine, -leucine, -methionine, -serine, and -tryptophan, 0.4 nmole each of lzCaspartic acid, -arginine, -glycine, isoleutine, -lysine, -phenylalanine, -proline, -threand -valine, and 0.2 onine, -tyrosine, PCi each of the latter ten 14C-labeled amino acids (all from New England Nuclear, at specific activities of 200-300 mCi/mmole). The final additions were 30 pg of calcium leucovorin (N-formyl tetrahydrofolate) and the desired RNA to program the system, generally about 250-400 pg. Incubation was for 30 minutes at 37”. The incorporation of 14C-amino acids into hot TCAinsoluble material was generally lO,OOO15,000 cpm per 25-J aliquot. The incubated reaction mixture was stored frozen until analysis by gel electrophoresis. Samples for electrophoresis were not centrifuged. Gel electrophoresis ancl autoradiography. The method of disc-electrophoresis in
200
WILHELM
AND
HASELKORN
sodium dodecyl sulfate (SDS) was used, This method was developed by Laemmli (1970) and the preparation of gels and of samples for electrophoresis followed his descriptions. Our separating gels were 10% acrylamide and the sample size was 50 ~1. Electrophoresis was carried out until the tracking dye had migrated 8.5 cm from the origin of the separating gel. The proteins were fixed by immersing the gel overnight in 10 % TCA. The gels were then rinsed with several changes of 5% TCA and 2 or 3 changes of 7.5% acetic acid. Autoradiograms were prepared by the method of Fairbanks et al. (1965). The dried gel slices were exposed to Kodak No Screen X-ray film for either 4 (in vivo patterns) or 8 days (in vitro).
P23
P24
:.”
24-
RESULTS
Gel Analysis
I
of Proteins Made in Vivo 0
I 2
I
I 4
I
I 6
I
I 8
Many phage proteins may be separated Migration Distance (cm) by the improved method of Laemmli (1970), which combines the resolution of FIG. 1. Patterns of proteins synthesized in cells discontinuous electrophoresis with the abil- infected with wild-type T4 and amber mutants in ity of SDS to dissociate proteins into indigenes 23 and 24. Cultures were pulse-labeled 20-24 Amounts of radioactivity vidual chains. Furthermore, this system min after infection. (cpm) applied to gels were 24-, 36,500; 23-, 35,600; separates proteins according to molecular wt., 12,000. weight as do continuous systems. Laemmli (1970) has identified the products of a number of T4 genes by comparing the gel One must note that these positions are patterns of radioactive proteins from cells those of the primary gene products, desiginfected with wild-type phage wit,h lysates nated as P23 (molecular weight, 56,000) and P24 (molecular weight, 45,000). In the infected with amber mutants in various genes. In the nonpermissive host, an amber course of normal assembly of heads in inmutation produces only a fragment of the fection with wild-type phage or tail-defecmutant protein; thus, one would expect tive mutants (but not with a number of the gel pattern obtained from such a lysate head-defective mutants) P23 is cleaved to to show either a protein band or altered yield a protein P23* (molecular weight, mobility or to completely lack a band, 46,000) which migrates in gels between depending on the size of the fragment. We P23 and P24 (Laemmli, 1970). P24 is also have confirmed Laemmli’s identification of cleaved, yielding P24* (43,500), which is some gene products and present our re- difficult to resolve from its precursor. The experiment of Fig. 2 shows the posisults as a reference to in vitro experiments. tion in gels of the product of gene 18 (molecThe data are presented as microdensitometer tracings from the autoradiograms of gels. ular weight, 69,000), the major component of the tail sheath (King, 1970). Also shown The product of gene 23 is the principal protein of the head (Sarabhai et al., 1964) are the positions of two of the largest proand that of gene 24 is one of the proteins teins made in infected cells, P34 and P37, required to determine the shape of the head the major components of the tail fiber (Ed(Laemmli et al., 1970). The positions of gar and Lielausis, 1965). These two proteins these proteins in the gel are shown in Fig. 1. have been assigned molecular weights,
IN VITRO SYNTHESIS P34
OF T4 PROTEINS:
201
GENE PRODUCTS
PI8 P 19
I P37 I
n
1 0
I
I 2
Migration
I
I 4 Distance
I
I 6
I
19-
I 8
(cm)
FIG. 2. Patterns of proteins synthesized in cells infected with T4 amber mutants in genes 37, 34, and 18. Pulse label as for Fig. 1. Amounts of radioactivity (cpm) applied to gells were: 37-, 31,400; 34-, 31,100; Es-, 29,500. respectively, of 160,000 and 114,000 (S. Ward, personal communication). An experiment which determines the positions of two of the smaller T4 proteins is shown in Fig. 3. One, the protein band of greatest mobility in these gels, is seen to correspond to the product of gene 19 (molecular weight, 18,000) which is thought to be the major protein of the tail tube (King, 1970). The other, a minor band of approximate molecular weight 25,000 (U. Laemmli, personal communication), is the product of gene 9. This protein acts in the assembly of the base plate and is likely to be incorporated into that structure (Flatgaard, 1969). In this experiment, the protein pattern of a gene 57-defective infection serves as the wild-type reference. This is justified as P57 is too small to be resolved in these gels (though it may be seen in the discontinuous gel system without SDS). P57 is made in vitro (Wilhelm and Haselkorn, 1969) and studies on it’ will be the subject of another communication.
I
0
I
I
I
I
4
2 Migration
I
I
6 Distance
I
8 (cm)
FIG. 3. Patterns of proteins synthesized in cells infected with T4 amber mutants in genes 19,9, and 57. Pulse label as for Fig. 1. Amounts of radioactivity (cpm) applied to gels were: 19-, 18,900; 9-, 41,000; 57-, 43,000.
Gel Analz&s
of Proteins Made in Vitro
RNA extracted from T4-infected cells may be used to program protein synthesis in a cell-free extract. The products of in vitro synthesis may be analyzed by electrophoresis in polyacrylamide gels and compared to proteins made in vivo. The results of such an experiment are presented in Fig. 4. There are striking similarities between the two gel patterns and some notable differences. The similarities observed are the basis for experiments described below on identification of specific in vitro proteins. The differences deserve further comment here. A major difference is the complete absence of the largest T4 proteins from the i?%vitro product. These include the products of genes 34 and 37, which are tail fiber components, and of gene 7, which is a base plate component. In addition the abundance of proteins in bands around the
202
WILHELM
AND HASELKORN
am+ 19 min RNA P23 1
I I
0
IP I
,
I
2 Migration
,
1
4
IP ,
,
6 Distance
I
8 (cm)
FIG. 4. Comparison of protein patterns from synthesis in vivo and in vitro. In vivo: Cells infected with an amber mutant in gene 57. (The gene 57 protein is too small to be resolved on these gels.) Pulse label as for Fig. 1. 43,000 cpm in proteins applied to gel. In vitro: Synthesis in 0.2 ml incubation with 150 pg of wild-type ,RNA isolated 19 min after infection. 26,000 cpm in proteins applied to the gel. position of the gene 18 products is much lower in vitro than in vivo. This is a reproducible result obtained with many different preparations of RNA. The discrimination appears to be based simply on size, since three different viral subassemblies whose genes are in three different regions of the T4 chromosome are among those whose products are affected. Other differences are due to the processing of viral capsid proteins in vivo (Laemmli, 1970). The most abundant protein in viva, P23*, is the cleavage product of the gene 23 protein, P23. Laemmli’s results suggest that the conversion P23 -+ P23* occurs only in a structure formed by the interaction of many gene products with T4 DNA. Since the conversion is assembly dependent, and it is unlikely that capsid assembly occurs in the in vitro system, we do not expect, to find P23* among the in vitro products. In fact, there is a weak band
near the position of P23* in the gels of in vitro products, but we shall show below that this band is the product of gene 24, rather than P23*. Another difference due to assemblydependent processing is in the band designated IP*. IP* is an internal protein released from T4 by osmotic shock; it too has a precursor, IP, whose cleavage requires conditions identical to those required for processing of P23. IP* is missing, as expected, from the in vitro products. There is an in vitro counterpart of the precursor, II?, but we cannot make a positive identification as no mutants which affect IP are available. Finally, there is one band in the in vitro pattern for which we have no explanation. It appears to be the most abundant in vitro product,, corresponding in molecular weight to approximately 35,000. This band appears among the products of syntheses directed by early and late T4 mRXA, and by RNA from uninfected E. coli. It is absent from syntheses to which mRNA is not added, from syntheses directed by RNA from SPOI-infected B. subtilis, and from syntheses directed by T4 Dl\jA in a coupled system. The IdentiJcation of Specific Products of in Vitro Synthesis Protein synthesis in vitro may be programmed with RNA isolated from cells infected with a given amber mutant of T4. Since this preparation contains a chainterminating codon within the message for a given protein, one may expect an alteration in the protein pattern, provided, of course, that the protein is made in vitro. Experiments of this type, therefore, indicate the fidelity of in vitro synthesis to the in vivo process. The protein patterns ob-
tained with RNAs from three amber infections and from wild-type T4 infection are compared in Fig. 5. The results demonstrate the presence among the in vitro products of three structural proteins, PM, the tail-sheath protein, P19, the tail-core protein, and 1’9, a base-plate protein. The experiment of Fig. 6 indicates that the in vitro proteins include the gene 24
IN in vitro
VI!!‘RO
SYNTHESIS
OF T4 PROTEINS:
203
GENE PRODUCTS
protein synthesis
am+ 19 min. RNA
MD 19 min.RNA
*------
DO 19 min.RNA
4 Distance
Migration
FIG. 5b. Expansion of portions of the patterns shown in Fig. 5a. I. Proteins directed by 1% and wild-type late RNA; vertical expansion achieved by a steeper optical wedge in the densitometer. II. Proteins directed by Q- and wild-type late RNA; horizontal expansion was 5X rather than the standard 2X.
DO Gmin. RNA
omt 6min.RNA
5 (cm)
,,,/
I(
\
P24 I
P 38
protein synthesis
0
2
Migration FIG.
5a. Patterns
4
8
6
Distance
of proteins
(cm)
synthesized
in
vitro with RNA from cells infected with wild-type T4 and umber mutants in genes 18, 19, and 9.
Synthesis in 0.4 ml incubations with 19 min RNA as follows: W, 300 pg; 19-, 520 pg; Q-, 320 pg; wt. (in 0.2 ml), 150 pg. Amounts of radioactivity (cpm) applied to gels were: 18-, 25,400; lQ-, 25,600; Q-, 22,400; wt., 26,000.
product, whose cleavage product is a component of the phage head (Laemmli, 1970), and the gene 38 product. This protein is involved in the assembly of tail fibers (Edgar and Lielausis, 1965; King and Wood, 1969), but it is not clear if it is incorporated into the final phage structure. The final experiment on the identification of in vitro proteins (Fig. 7) demonstrates thar P23, the precursor to the major head protein, can be made in vitro. The experiment also shows the position in the gel system of the
\ I I 0
I
2 Migration
I
I
4
I
I
6
I
I
8
Distance(cm)
FIG. 6. Patterns of proteins synthesized in vitro with RNA from cells infected with umber mutants in genes 24 and 38. Protein synthesis in 0.4 ml incubations with 19 min RNA from 24-, 330 pg, or 38-, 410 pg. Amounts of radioactivity (cpm) applied to gels were: 24-, 29,200; 38-, 26,500.
204
WILHELM
in vitro
AND HASELKORN
protein synthesis P23
in vitro
protein
synthesis
P22 am+ 19min. RNA n
am+ Gmin. RNA
I
I
L I I
0
I
I
2 Migration
I
I
I
,
4 6 Distance (cm)
I
8
FIG. 7. Patterns of proteins synthesized in vitro with RNA from cells infected with umber mutants in genes 22 and 23. Protein synthesis in 0.2 ml incubations with 19 min RNA from 22-, 225 pg, or 23-, 255 pg. Amounts of radioactivity (cpm) applied to the gels were: 22-, 26,200; 23-, 18,000.
gene 22 product (mol. wt. 31,000; Laemmli, 1970). We have previously demonstrated in vitro synthesis of this protein and in vitro suppression of an amber mutation (Wilhelm and Haselkorn, 1969). It is interesting to note that P22 is also cleaved in wivo; the products are not found on the gels of in vitro protein synthesis or of extracts prepared in vivo. Gel Analysis of Proteins Made in Vitro with Early RNA and RNA from Pleiotropic Mutants of T4 In the experiments above, the RNAs used to program protein synthesis were all isolated late in infection, that is, 19 min at 30”. We may also ask what proteins are synthesized with RNA isolated at an early time (6 min) after infection. The results of this experiment are given in Fig. 8; there are a number of proteins made, but they do not correspond to the structural proteins P18, P23, P24, P22, P9, P38, or P19. RNA was also prepared early and late from infection of the nonpermissive host
0
8 Migration
Distance
(cm)
FIG. 8. Patterns of proteins synthesized in vitro with RNA isolated from cells early and late after T4 wild-type infection. Protein synthesis programmed with 150 pg of 19 min RNA (0.2 ml incubation) or 26Oog of 6 min RNA (0.4 ml incubation). Amounts of radioactivity applied to gels were 19 min, 26,000; 6 min, 33,600.
(E. coli BE) with a T4 mutant defective in DNA replication (gene 45) and the gene 55 mutant (MD, for maturation defective), which allows DNA replication but does not make phage structures. The results of this experiment (Fig. 9) indicate that both types of RNA, even isolated at 19 min, do not direct the synthesis of discernible amounts of those structural gene products which we know to be synthesized in vitro. Thus, for each of these gene products, the absence of the protein late in DO or MD infection can be accounted for by the absence of message. This result agrees with earlier conclusions drawn from DNARNA hybridization-competition experiments (Bolle et al., 1968b). The gel patterns shown in Fig. 9 provide some information beyond that already available from hybridization experiments. The shut off of early protein synthesis, characteristic of wild-type T4 infection, is delayed in DO mutant infection (Wiberg et al., 1962; Hosoda and Levinthal, 1968).
IN /n v/fro
VZTROSYNTHESIS
OF T4 PROTEINS: in vitro
protein synthesis
il
205
GENE PRODUCTS protein
synthesis
omt 19 min. RNA T4 ri bosomes
Uninfected cell ribosomes
1
-2-J-J 10
4
Migration
8
6
Distance
(cm)
FIG. 10. Patterns of proteins synthesized in vitro with ribosomes from uninfected and from T4infected cells. “T4 ribosomes” were isolated from E. coli BE infected for 13 min (37”) with amM41 (lysozyme). Otherwise protein synthesis was performed in the usual manner with 300 pg of 19 min wild-type RNA (0.4 ml incubation). Amounts of radioactivity (cpm) in proteirls applied to gels were: top tracing, 33,000; bottom tracing, 26,000.
The messages for bands A, B, and C appear to be absent late in gene 55 infection. The remainder of the gel pattern of Migration Distance (cm) proteins directed by 19 min RNA from FIG. 9. Patterns of proteins synthesized in vitro with RNAs from cells infected with wild-type and gene 55 infection is similar to the typical with umber mutants in gene 45 (DO) and gene 55 early pattern. This result leads to a further subdivision of early messages; those for (MD). Protein synthesis in 0.4 ml incubations programmed with RNA as follows: wt., 6 min, 260 bands A and C are absent late in wild pg; DO, 6 min, 340 rg; DO, 19 min, 400 pg; MD, type or gene 55 infection, but present late 19 min, 410rg; wt., 19 min (in 0.2 ml incubation), in DO infection. 150 pg. Amounts of radioactivity (cpm) applied to Several T4 proteins are firmly bound to the gels were: wt., 6 min, 33,600; DO, 6 min, 22,800; ribosomes in T4-infected cells (Smith and DO, 19 min, 30,000; MD, 19 min, 28,600; wt., 19 Haselkorn, 1969) and such ribosomes conmin, 26,000. tain factors that discriminate against MS2 RNA in vitro (Hsu and Weiss, 1969). NeverThe bands labeled A and C show this theless, substitution of ribosomes prepared property; their messages are present early 13 min after T4 infection of E. coli B at in wild-type or DO infection, absent late 37” for the ribosomes from uninfected cells in wild-type infection, but present late in the protein-synthesizing system does in DO infection. On the other hand, the not change the pattern of proteins synmessage for band B, present early in DO thesized in vitro directed by late T4 mRNA infection, is absent late in DO infection. (Fig. 10). The abundance distribution in Therefore it becomes necessary to divide the two cases is strikingly similar, including early messages into two subclasses with respect to their presence or absence late the large band at 35,000 mol. wt. whose origin is unknown. in DO infection. 2
4
6
8
206
WILHELM
AND
DISCUSSION
Our experiments demonstrate that a variety of structural proteins of T4, encompassing those of head, tail, and baseplate, may be synthesized in vitro with RNA from infected cells. A striking exception is the failure to synthesize the major proteins of the tail fibers, gene products 34 and 37, which are two of the largest phage proteins. Several possible explanations for this failure may be advanced. First, since the messages are necessarily large, they may be more susceptible to nuclease destruction during preparation than the smaller messages. This possibility is diicult to eliminate; however, the consistent failure of some twenty RNA preparations to make these proteins suggests something more basic. The genes involved in tail-fiber formation, genes 34-38, form a cluster which is displaced from the rest of the “late” region of the T4 genome. It is conceivable that the translation of the entire group requires some modification not present in the proteinsynthesizing systems from uninfected cells. Two observations make this explanation unlikely: (1) the product of one gene in the cluster, gene 38, of molecular weight approximately 25,000, can be made in such extracts in vitro; (2) the use of ribosomes or supernatant proteins from T4-infected cells in the system does not permit the synthesis of proteins 34 and 37 (J.W., unpublished observation). A third explanation, which we consider more likely, is based on the nature of the mRNA isolated. Morse et al. (1969) found that, upon de-repression of the tryptophan operon in E. coli, a wave of message synthesis occurs which is closely coupled to translation by a closely spaced cluster of ribosomes. This cluster is followed by message degradation from the 5’-end, which begins well before transcription of the operon has reached the 3’-end. Morse et al. calculated that the cluster of ribosomes would encompass a message length of some 20004000 nucleotides; this might be the length of message protected from nuclease action. The T4 gene products 37 and 34, of molecular weights 114,000 and 166,000, require
HASELKORN
messages of 3000-5000 nucleotides. Therefore, if the coupling of transcription, translation, and degradation in T4-infected cells is analogous to that observed with the tryptophan operon, functionally intact messengers for the uncoupled synthesis of these proteins in vitro simply may not be obtainable from infected cells. Our experiments answer some questions about the kind of RNA required for synthesis of the structural proteins. It is clear that RNA populations from cells early in infection are not effective; synthesis of these proteins is achieved only with RNA taken during the period when the infected cells are making the “late” RNA defined by hybridization-competition to experiments (Bolle et al., 1968a). In addition, in the course of infection of the nonpermissive host with DNA-negative mutants or with mutants in gene 55, little or no late messenger is ever made (Bolle et al., 196Sb). In agreement, we find that populations of RNA made late in DO or gene 55 infection do not direct the synthesis of the late, structural proteins. We note further that the in vitro synthesis of T4-specific proteins occurs in cell-free extracts derived from uninfected cells. This observation, plus the correlation of late message with the synthesis of late proteins, lends further support to the concept that the control of T4 development is largely transcriptional, and that translational modifications play no major, positive, roles. The transcription of early messages in T4 infection is under both positive and negative control. When cells are infected in the presence of chloramphenicol, only immediate early species of RNA are made. Delayed early species require the expression of one or more immediate early genes for their transcription (Salser et al., 1970; Milanesi et al., 1970). The internal proteins are made in vitro by chloramphenicol RNA (L. RI. Black, Jr. and L. Gold, personal communication). We find only a few one correspondmg in bands, including position to the largest internal protein, directed by chloramphenicol RNA (J. M. W., unpublished). The majority of bands whose synthesis is directed by early RNA
IN
VITRO
SYNTHESIS
OF T4 PROTEINS
are delayed early proteins. Among these, there appear to be three modes of negative control. The shut off of one class requires DNA replication and gene 55 function. Another class is shut off without gene 55, and a third, represented by band B in Fig. 9, is shut off even in the absence of DNA replication. ACKNOWLEDGMENTS This research was supported by a grant from the National Institutes of Health (AI-GM-09315). One of us (J.M.W.) was the recipient of a postdoctoral fellowship from the American Cancer Society. We t.hank 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. (1968a). Transcription during bacteriophage T4 development: Synthesis and relative stability of early and late RNA. J. Mol. Biol. 31, 325-348. BOLLE, A., EPSTEIN, R. H., S~LS~R, W., and GEIDUSCHEK, E. P. (1968b). Transcription during bacteriophage T4 development : Requirements for late messenger synthesis. J. Mol. Biol. 33, 339362. COOLSM~, J., and HASELKORN, R. (1969). In vitro synthesis of T4 proteins. Biochem. Biophys. Res. Commun 34, 253-259. EDGAR, R. S., and LIEL~USIS, I. (1965). Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52, 1187-1200. EPSTEIN, R. H., BOLLE, A., STEINRF:RO, C. M., KELLENBERGER, E., BOY DF, L.* TOUR, E., CH~:VALLEY, R., EDGAR, R. S., SUSM~N, M., DENHARDT, G. H., and LIsLaUSIS, I. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. FAIRBANKS, G., JR., LEVINTHAL, C., and REF,DER, 1~. H. (1965). Analysis of X-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393-399. FLATGA.~RD, J. (1969). The role of the gene 9 product in the assembly and triggering of bacteriophage T4. Thesis. California Institute of Technology. GOLD, L. M., and SCHWEIGER, M. (1969). Synthesis of phage-specific a- and @glucosyl transferases directed by T-even DNA in vitro. Proc. Nat. Acacl. Sci. U.S. 62,892X398.
: GENE
PRODUCTS
207
HOSOD~, J., and LEVINTHAL, C. (1968). Protein synthesis by E. coli infected with bact,eriophage T4D. Virology 34,709-727. Hsu, W. T., and WEISS, S. B. (1969). Selective translation of T4 template RNA by ribosomes from T4-infected E. coli. Proc. Nat. Acad. Sci. u. s. 64,345-351. KING, J. (1968). Assembly of the t,ail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J. (1970). In vilro assembly of t.he bacteriophage T4 tail core: three sequential steps. Fed. Eur. Biochem. Sot. Symp. 21, 156. KING, J., and WOOD, W. B. (1969). Assembly of bacteriophage T4 t,ail fibers: the sequence of gene product interaction. J. fifol. Biol. 39, 503601. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. iVuture (London) 227, 680685. LAEMMLI, U. K., MOLBI~:RT, E., SHOUX, M., and KELLENBERGEH, E. (1970). Form-determining fun&ion of the genes required for the assembly of the head of bacteriophage T4. J. Mol. Biol. 49,99-114. MILANESI, G., BRODY, E. N., GRSU, O., and GEIDUSCHEK, E. P. (1970). Transcription of the bacteriophage T4 template in vitro: Separation of “Delayed Early” from “Immediate Early” transcript.ion. Proc. i\rat. Acod. Sci. U. S. 66, 181-188. MORSE, D. E. MOSTF:LLER, R. D., and Y.INOFSICY, C. (1969). Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spring Harbor Symp. Quant. Biol. 34, 725-740. R. (1970). SALSF,R, W., BOLLE, A., and EPSTEIN, Transcription during bacteriophage T4 development : A demonstration that distinct subclasses of “early” RNA appear at different times and that some are “turned off” at late t,imes. J. Mol. Biol. 49, 271-296. SARIBHAI, A. S., STRETTON, A. 0. W., BRENNER, S., and BOLLE, A. (1964). Co-linearity of the gene with the polypeptide chain. Nature (London) 201, 13-17. SCHW&XGER, M., and GOLD, L. M. (1969). Bact)eriophage T4 DNA-dependent in vitro synthesis of lysozyme. Proc. Nat. Acad. Sci. U. S. 63, 1351-1358. SMITH, F. L., and HAS~LKORN, IX.. (1969). Proteins associated with ribosomes in T4-infected E. coli Cold Spring Harbor Symp. Quant. Biol. 34, 91-94. WIBF,RG, J. S., DIRKSI”N, M., EPSTEIN, R. H., LURID, S. E., and &JCHANAN, J. M. (1962). Early enzyme synthesis and its control in E.
208
WILHELM
AND
coli infected with some amber mutants of bacteriophage T4. Proc. Nat. Acad. Sci. U. S. 48,293-302. 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. Quant. BioZ. 34, 793-798.
HASELKORN YOUNG, E. T. (1970). Cell-free synthesis of bacteriophage T4 glucosyl transferase. J. Mol. Biol. 51, 591-604. YOUNG, E. T., II, and VAN HOTJWE, G. (1970). Control of synthesis of glucosyl transferase and lysozyme messengers after T4 infection. J. Mol. BioZ. 51, 605-620.
43, 198-208 (1971)
In Vitro
Synthesis Genes
JAMES Department
of T4 Proteins:
The Products
9, 18, 19, 23, 24, and
M. WILHELM’ of Biophysics,
ROBERT
AND
38
HASELKORN
of Chicago,
University
of
Chicago,
Illinois
60637
Accepted September 28, 1970 The improved acrylamide gel electrophoresis procedure of Laemmli (1970) permits identification of a number of specific T4 gene products among the proteins whose synthesis is directed in vitro by RNA from T4-infected E. coli. RNA isolated late in infection directs the synthesis of the products of genes 9 (baseplate), 18 (tail sheath), 19 (tail core), 23 and 24 (head), and 38 (tail fiber). Synthesis of these proteins is not directed by RNA isolated early in wild-type T4 infection, or by RNA isolated late in DNA-negative or maturation defective infection. These results are consistent with the suggestion that T4 gene expression is regulated primarily at the level of transcription. A major difference between the products of in vitro protein synthesis and the display of proteins made in viva is the absence of proteins of molecular weight greater than 80,000 from the former. We believe this result is a consequence of transcription-translation coupling in wivo, which limits the size of functional messages that can be isolated. The proteins made in vitro also lack P23*, a protein found in viva as a result of “processing” of the primary product of gene 23. INTRODUCTION
The synthesis of specific proteins in vitro is a useful tool for the analysis of populations of messenger RNA. The technique has been applied most extensively to RNA from Escherichia coli infected with bacteriophage T4, with which the in vitro synthesis of enzymatically active lysozyme (Salser et al., 1967) and the G+ and fl-glucosyl transferases (Young, 1970; Young and Van Houwe, 1970) has been demonstrated. It. is also possible to couple transcription and translation in vitro to direct the synthesis of these activities with T4 DNA (Gold and Schweiger, 1969; Schweiger and Gold, 1969). There are many T4 proteins, especially the structural components of the phage, which have no readily demonstrable enzymatic activities. The synthesis of these gene products in infected cells may be studied by electrophoresis of labeled cell extracts in polyacrylamide gels (Hosoda and i Present address : Department biology, University of Pennsylvania, phia, Pennsylvania.
of
Micro-
Philadel-
Levinthal, 1968). We have used gel electrophoresis to examine t,he products of T4 mRNA-dependent protein synthesis (Coolsma and Haselkorn, 1969) and have reported the specific synthesis of the products of T4 genes 22 and 57 (Wilhelm and Haselkorn, 1969). The gene 22 product is a factor in the control of the shape of the phage head (Laemmli et al., 1970) and gene 57 pIays a regulatory role in the formation of tail fibers (Edgar and Lielausis, 1965), most likely at the level of assembly (S. Ward, personal communication). In the present communication we report the in vitro synthesis
of
a variety
of
other
proteins of T4 directed by RNA from cells after phage infection. MATERIALS
AND
structural
isolated
METHODS
Bacteria and bacteriophages. E. coli BE, the nonpermissive host, was used for preparat’ion of protein-synthesis systems and for all infected-cell RNAs. Stocks of amber phages were grown on E. coli CR63 (Sul+). The amber mutants of T4 have been de-
198
lAr VZTRO
SYNTHESIS
OF T4 PROTEINS:
scribed (Epstein et al., 1963) and are listed here so that they may be referred to later only by gene number: gene 9 defective (9-), arnE17; lo-, B255; 18-, EM; 19-, KG524; 22-, N98; 23-, B17, 24-, E355; 34-, a double mutant, B25-A455; 37-, a double mutant, N52-B280; 3S-, B262; 45-, ElO; 55-, BL292; 57-, E198. Media. For lysates and RNA: M9, which contains, per liter, 7 g Na2HP04. 2Hz0, 3 g KH2P04, 0.5 g NaCI, 1 g NH&I, 1O-3 d1 MgSOd, lop4 M CaC&, and 4 g glucose was used. 119s is the same supplemented with 10 g Casamino acids (Difco). For growth of cells for protein-synthesizing extracts: per liter, 10 g Bactotryptone and 1 g yeast extract (Difco), 8 g NaCl, and 1 g glucose was used. Preparation of radioactive lysafes. E. coli BE were grown to a density of 4 X lo8 cells/ml in Sl9 at 37”, shifted to 30” and supplemented with n-tryptophan (40 pg/ ml). 5 ml of culture were infected at a m.o.i. = 5 and at 20 min after infection the proteins were labeled by addition of 1.25 &i of leucine-14C (315 mCi/mmole). Four minutes later the culture was chilled with ice, the cells collected by centrifugation and the cell pellet was resuspended in 0.5 ml of 0.01 M Tris.HCl, pH 7.6, 0.01 M I\Ig acetate. This suspension was stored frozen. The incorporation of leucine-14C into hot trichloroacetic acid (TCA) -insoluble material was generally 3000-5000 cpm (Beckman Low Beta Gas-flow Counter; efficiency 17%) in a 50-~1 aliquot of culture. Preparation of RI+‘A from infected cells. E. coli BE were grown in M9S to a density of 5 X 10e cells/ml at 30”, supplemented and infected as dewith n-tryptophan, scribed. The RNA was isolated as described by Bolle et al. (1968a) with the following modifications: egg white lysozyme was added to the resuspended cells only when RNA was prepared at 6 minutes after infection or when no endogenous lysozyme is produced (infection with gene 45 or gene 55 mutants) ; the first phenol extraction was performed at 65” for 2 min but the two subsequent extractions were at 37”; after ethanol precipitation, the RNA was resuspended in 0.01 M Tris .HCl, pH 7.6, to an approximate concentration of 5
GENE
PRODUCTS
199
mg/ml and dialyzed against distilled water for 6 hr in the cold. Protein synthesis. Cells were grown in the medium given above at 37” until the turbidity reached 0.7 at 650 nm. The cells were chilled with ice and collected by centrifugation. The cell pellet was washed twice by resuspension in 0.01 M Tris.HCl, pH 7.6, 0.01 M lllg acetate and centrifugation, then frozen and stored overnight. Cell-free extract was prepared and separated into ribosomes and a supernatant protein fraction by the method of Gold and Schweiger (1969) with two modifications: the cells were broken by grinding with alumina (2.5 X the wet weight of cells) and the buffer used for the extraction of the paste of cells and alumina contained pancreatic deoxyribonuclease (Worthington, electrophoretically pure) at 1 pg/ml. In vitro protein synthesis was generally carried out in an incubation volume of 0.4 ml. This contained 1.4 mg ribosomes, 600 pg of supernatant protein fraction, 400 pg of E. coli B transfer RNA (General Biochemicals), 30 pmoles Tris. HCl, pH 7.6, 10 pmoles KCl, 28 pmoles NH&I; 3.4 pmoles MgSO+ 0.5 pmole dithiothreitol, 2 pmoles phosphoenol pyruvate, 5 pg pyruvate kinase, 1.2 pmoles ATP, 0.08 pmole GTP, 6 nmoles each of ‘Y-alanine, -asparagine, -cysteine, -glutamic acid, -glutamine, -histidine, -leucine, -methionine, -serine, and -tryptophan, 0.4 nmole each of lzCaspartic acid, -arginine, -glycine, isoleutine, -lysine, -phenylalanine, -proline, -threand -valine, and 0.2 onine, -tyrosine, PCi each of the latter ten 14C-labeled amino acids (all from New England Nuclear, at specific activities of 200-300 mCi/mmole). The final additions were 30 pg of calcium leucovorin (N-formyl tetrahydrofolate) and the desired RNA to program the system, generally about 250-400 pg. Incubation was for 30 minutes at 37”. The incorporation of 14C-amino acids into hot TCAinsoluble material was generally lO,OOO15,000 cpm per 25-J aliquot. The incubated reaction mixture was stored frozen until analysis by gel electrophoresis. Samples for electrophoresis were not centrifuged. Gel electrophoresis ancl autoradiography. The method of disc-electrophoresis in
200
WILHELM
AND
HASELKORN
sodium dodecyl sulfate (SDS) was used, This method was developed by Laemmli (1970) and the preparation of gels and of samples for electrophoresis followed his descriptions. Our separating gels were 10% acrylamide and the sample size was 50 ~1. Electrophoresis was carried out until the tracking dye had migrated 8.5 cm from the origin of the separating gel. The proteins were fixed by immersing the gel overnight in 10 % TCA. The gels were then rinsed with several changes of 5% TCA and 2 or 3 changes of 7.5% acetic acid. Autoradiograms were prepared by the method of Fairbanks et al. (1965). The dried gel slices were exposed to Kodak No Screen X-ray film for either 4 (in vivo patterns) or 8 days (in vitro).
P23
P24
:.”
24-
RESULTS
Gel Analysis
I
of Proteins Made in Vivo 0
I 2
I
I 4
I
I 6
I
I 8
Many phage proteins may be separated Migration Distance (cm) by the improved method of Laemmli (1970), which combines the resolution of FIG. 1. Patterns of proteins synthesized in cells discontinuous electrophoresis with the abil- infected with wild-type T4 and amber mutants in ity of SDS to dissociate proteins into indigenes 23 and 24. Cultures were pulse-labeled 20-24 Amounts of radioactivity vidual chains. Furthermore, this system min after infection. (cpm) applied to gels were 24-, 36,500; 23-, 35,600; separates proteins according to molecular wt., 12,000. weight as do continuous systems. Laemmli (1970) has identified the products of a number of T4 genes by comparing the gel One must note that these positions are patterns of radioactive proteins from cells those of the primary gene products, desiginfected with wild-type phage wit,h lysates nated as P23 (molecular weight, 56,000) and P24 (molecular weight, 45,000). In the infected with amber mutants in various genes. In the nonpermissive host, an amber course of normal assembly of heads in inmutation produces only a fragment of the fection with wild-type phage or tail-defecmutant protein; thus, one would expect tive mutants (but not with a number of the gel pattern obtained from such a lysate head-defective mutants) P23 is cleaved to to show either a protein band or altered yield a protein P23* (molecular weight, mobility or to completely lack a band, 46,000) which migrates in gels between depending on the size of the fragment. We P23 and P24 (Laemmli, 1970). P24 is also have confirmed Laemmli’s identification of cleaved, yielding P24* (43,500), which is some gene products and present our re- difficult to resolve from its precursor. The experiment of Fig. 2 shows the posisults as a reference to in vitro experiments. tion in gels of the product of gene 18 (molecThe data are presented as microdensitometer tracings from the autoradiograms of gels. ular weight, 69,000), the major component of the tail sheath (King, 1970). Also shown The product of gene 23 is the principal protein of the head (Sarabhai et al., 1964) are the positions of two of the largest proand that of gene 24 is one of the proteins teins made in infected cells, P34 and P37, required to determine the shape of the head the major components of the tail fiber (Ed(Laemmli et al., 1970). The positions of gar and Lielausis, 1965). These two proteins these proteins in the gel are shown in Fig. 1. have been assigned molecular weights,
IN VITRO SYNTHESIS P34
OF T4 PROTEINS:
201
GENE PRODUCTS
PI8 P 19
I P37 I
n
1 0
I
I 2
Migration
I
I 4 Distance
I
I 6
I
19-
I 8
(cm)
FIG. 2. Patterns of proteins synthesized in cells infected with T4 amber mutants in genes 37, 34, and 18. Pulse label as for Fig. 1. Amounts of radioactivity (cpm) applied to gells were: 37-, 31,400; 34-, 31,100; Es-, 29,500. respectively, of 160,000 and 114,000 (S. Ward, personal communication). An experiment which determines the positions of two of the smaller T4 proteins is shown in Fig. 3. One, the protein band of greatest mobility in these gels, is seen to correspond to the product of gene 19 (molecular weight, 18,000) which is thought to be the major protein of the tail tube (King, 1970). The other, a minor band of approximate molecular weight 25,000 (U. Laemmli, personal communication), is the product of gene 9. This protein acts in the assembly of the base plate and is likely to be incorporated into that structure (Flatgaard, 1969). In this experiment, the protein pattern of a gene 57-defective infection serves as the wild-type reference. This is justified as P57 is too small to be resolved in these gels (though it may be seen in the discontinuous gel system without SDS). P57 is made in vitro (Wilhelm and Haselkorn, 1969) and studies on it’ will be the subject of another communication.
I
0
I
I
I
I
4
2 Migration
I
I
6 Distance
I
8 (cm)
FIG. 3. Patterns of proteins synthesized in cells infected with T4 amber mutants in genes 19,9, and 57. Pulse label as for Fig. 1. Amounts of radioactivity (cpm) applied to gels were: 19-, 18,900; 9-, 41,000; 57-, 43,000.
Gel Analz&s
of Proteins Made in Vitro
RNA extracted from T4-infected cells may be used to program protein synthesis in a cell-free extract. The products of in vitro synthesis may be analyzed by electrophoresis in polyacrylamide gels and compared to proteins made in vivo. The results of such an experiment are presented in Fig. 4. There are striking similarities between the two gel patterns and some notable differences. The similarities observed are the basis for experiments described below on identification of specific in vitro proteins. The differences deserve further comment here. A major difference is the complete absence of the largest T4 proteins from the i?%vitro product. These include the products of genes 34 and 37, which are tail fiber components, and of gene 7, which is a base plate component. In addition the abundance of proteins in bands around the
202
WILHELM
AND HASELKORN
am+ 19 min RNA P23 1
I I
0
IP I
,
I
2 Migration
,
1
4
IP ,
,
6 Distance
I
8 (cm)
FIG. 4. Comparison of protein patterns from synthesis in vivo and in vitro. In vivo: Cells infected with an amber mutant in gene 57. (The gene 57 protein is too small to be resolved on these gels.) Pulse label as for Fig. 1. 43,000 cpm in proteins applied to gel. In vitro: Synthesis in 0.2 ml incubation with 150 pg of wild-type ,RNA isolated 19 min after infection. 26,000 cpm in proteins applied to the gel. position of the gene 18 products is much lower in vitro than in vivo. This is a reproducible result obtained with many different preparations of RNA. The discrimination appears to be based simply on size, since three different viral subassemblies whose genes are in three different regions of the T4 chromosome are among those whose products are affected. Other differences are due to the processing of viral capsid proteins in vivo (Laemmli, 1970). The most abundant protein in viva, P23*, is the cleavage product of the gene 23 protein, P23. Laemmli’s results suggest that the conversion P23 -+ P23* occurs only in a structure formed by the interaction of many gene products with T4 DNA. Since the conversion is assembly dependent, and it is unlikely that capsid assembly occurs in the in vitro system, we do not expect, to find P23* among the in vitro products. In fact, there is a weak band
near the position of P23* in the gels of in vitro products, but we shall show below that this band is the product of gene 24, rather than P23*. Another difference due to assemblydependent processing is in the band designated IP*. IP* is an internal protein released from T4 by osmotic shock; it too has a precursor, IP, whose cleavage requires conditions identical to those required for processing of P23. IP* is missing, as expected, from the in vitro products. There is an in vitro counterpart of the precursor, II?, but we cannot make a positive identification as no mutants which affect IP are available. Finally, there is one band in the in vitro pattern for which we have no explanation. It appears to be the most abundant in vitro product,, corresponding in molecular weight to approximately 35,000. This band appears among the products of syntheses directed by early and late T4 mRXA, and by RNA from uninfected E. coli. It is absent from syntheses to which mRNA is not added, from syntheses directed by RNA from SPOI-infected B. subtilis, and from syntheses directed by T4 Dl\jA in a coupled system. The IdentiJcation of Specific Products of in Vitro Synthesis Protein synthesis in vitro may be programmed with RNA isolated from cells infected with a given amber mutant of T4. Since this preparation contains a chainterminating codon within the message for a given protein, one may expect an alteration in the protein pattern, provided, of course, that the protein is made in vitro. Experiments of this type, therefore, indicate the fidelity of in vitro synthesis to the in vivo process. The protein patterns ob-
tained with RNAs from three amber infections and from wild-type T4 infection are compared in Fig. 5. The results demonstrate the presence among the in vitro products of three structural proteins, PM, the tail-sheath protein, P19, the tail-core protein, and 1’9, a base-plate protein. The experiment of Fig. 6 indicates that the in vitro proteins include the gene 24
IN in vitro
VI!!‘RO
SYNTHESIS
OF T4 PROTEINS:
203
GENE PRODUCTS
protein synthesis
am+ 19 min. RNA
MD 19 min.RNA
*------
DO 19 min.RNA
4 Distance
Migration
FIG. 5b. Expansion of portions of the patterns shown in Fig. 5a. I. Proteins directed by 1% and wild-type late RNA; vertical expansion achieved by a steeper optical wedge in the densitometer. II. Proteins directed by Q- and wild-type late RNA; horizontal expansion was 5X rather than the standard 2X.
DO Gmin. RNA
omt 6min.RNA
5 (cm)
,,,/
I(
\
P24 I
P 38
protein synthesis
0
2
Migration FIG.
5a. Patterns
4
8
6
Distance
of proteins
(cm)
synthesized
in
vitro with RNA from cells infected with wild-type T4 and umber mutants in genes 18, 19, and 9.
Synthesis in 0.4 ml incubations with 19 min RNA as follows: W, 300 pg; 19-, 520 pg; Q-, 320 pg; wt. (in 0.2 ml), 150 pg. Amounts of radioactivity (cpm) applied to gels were: 18-, 25,400; lQ-, 25,600; Q-, 22,400; wt., 26,000.
product, whose cleavage product is a component of the phage head (Laemmli, 1970), and the gene 38 product. This protein is involved in the assembly of tail fibers (Edgar and Lielausis, 1965; King and Wood, 1969), but it is not clear if it is incorporated into the final phage structure. The final experiment on the identification of in vitro proteins (Fig. 7) demonstrates thar P23, the precursor to the major head protein, can be made in vitro. The experiment also shows the position in the gel system of the
\ I I 0
I
2 Migration
I
I
4
I
I
6
I
I
8
Distance(cm)
FIG. 6. Patterns of proteins synthesized in vitro with RNA from cells infected with umber mutants in genes 24 and 38. Protein synthesis in 0.4 ml incubations with 19 min RNA from 24-, 330 pg, or 38-, 410 pg. Amounts of radioactivity (cpm) applied to gels were: 24-, 29,200; 38-, 26,500.
204
WILHELM
in vitro
AND HASELKORN
protein synthesis P23
in vitro
protein
synthesis
P22 am+ 19min. RNA n
am+ Gmin. RNA
I
I
L I I
0
I
I
2 Migration
I
I
I
,
4 6 Distance (cm)
I
8
FIG. 7. Patterns of proteins synthesized in vitro with RNA from cells infected with umber mutants in genes 22 and 23. Protein synthesis in 0.2 ml incubations with 19 min RNA from 22-, 225 pg, or 23-, 255 pg. Amounts of radioactivity (cpm) applied to the gels were: 22-, 26,200; 23-, 18,000.
gene 22 product (mol. wt. 31,000; Laemmli, 1970). We have previously demonstrated in vitro synthesis of this protein and in vitro suppression of an amber mutation (Wilhelm and Haselkorn, 1969). It is interesting to note that P22 is also cleaved in wivo; the products are not found on the gels of in vitro protein synthesis or of extracts prepared in vivo. Gel Analysis of Proteins Made in Vitro with Early RNA and RNA from Pleiotropic Mutants of T4 In the experiments above, the RNAs used to program protein synthesis were all isolated late in infection, that is, 19 min at 30”. We may also ask what proteins are synthesized with RNA isolated at an early time (6 min) after infection. The results of this experiment are given in Fig. 8; there are a number of proteins made, but they do not correspond to the structural proteins P18, P23, P24, P22, P9, P38, or P19. RNA was also prepared early and late from infection of the nonpermissive host
0
8 Migration
Distance
(cm)
FIG. 8. Patterns of proteins synthesized in vitro with RNA isolated from cells early and late after T4 wild-type infection. Protein synthesis programmed with 150 pg of 19 min RNA (0.2 ml incubation) or 26Oog of 6 min RNA (0.4 ml incubation). Amounts of radioactivity applied to gels were 19 min, 26,000; 6 min, 33,600.
(E. coli BE) with a T4 mutant defective in DNA replication (gene 45) and the gene 55 mutant (MD, for maturation defective), which allows DNA replication but does not make phage structures. The results of this experiment (Fig. 9) indicate that both types of RNA, even isolated at 19 min, do not direct the synthesis of discernible amounts of those structural gene products which we know to be synthesized in vitro. Thus, for each of these gene products, the absence of the protein late in DO or MD infection can be accounted for by the absence of message. This result agrees with earlier conclusions drawn from DNARNA hybridization-competition experiments (Bolle et al., 1968b). The gel patterns shown in Fig. 9 provide some information beyond that already available from hybridization experiments. The shut off of early protein synthesis, characteristic of wild-type T4 infection, is delayed in DO mutant infection (Wiberg et al., 1962; Hosoda and Levinthal, 1968).
IN /n v/fro
VZTROSYNTHESIS
OF T4 PROTEINS: in vitro
protein synthesis
il
205
GENE PRODUCTS protein
synthesis
omt 19 min. RNA T4 ri bosomes
Uninfected cell ribosomes
1
-2-J-J 10
4
Migration
8
6
Distance
(cm)
FIG. 10. Patterns of proteins synthesized in vitro with ribosomes from uninfected and from T4infected cells. “T4 ribosomes” were isolated from E. coli BE infected for 13 min (37”) with amM41 (lysozyme). Otherwise protein synthesis was performed in the usual manner with 300 pg of 19 min wild-type RNA (0.4 ml incubation). Amounts of radioactivity (cpm) in proteirls applied to gels were: top tracing, 33,000; bottom tracing, 26,000.
The messages for bands A, B, and C appear to be absent late in gene 55 infection. The remainder of the gel pattern of Migration Distance (cm) proteins directed by 19 min RNA from FIG. 9. Patterns of proteins synthesized in vitro with RNAs from cells infected with wild-type and gene 55 infection is similar to the typical with umber mutants in gene 45 (DO) and gene 55 early pattern. This result leads to a further subdivision of early messages; those for (MD). Protein synthesis in 0.4 ml incubations programmed with RNA as follows: wt., 6 min, 260 bands A and C are absent late in wild pg; DO, 6 min, 340 rg; DO, 19 min, 400 pg; MD, type or gene 55 infection, but present late 19 min, 410rg; wt., 19 min (in 0.2 ml incubation), in DO infection. 150 pg. Amounts of radioactivity (cpm) applied to Several T4 proteins are firmly bound to the gels were: wt., 6 min, 33,600; DO, 6 min, 22,800; ribosomes in T4-infected cells (Smith and DO, 19 min, 30,000; MD, 19 min, 28,600; wt., 19 Haselkorn, 1969) and such ribosomes conmin, 26,000. tain factors that discriminate against MS2 RNA in vitro (Hsu and Weiss, 1969). NeverThe bands labeled A and C show this theless, substitution of ribosomes prepared property; their messages are present early 13 min after T4 infection of E. coli B at in wild-type or DO infection, absent late 37” for the ribosomes from uninfected cells in wild-type infection, but present late in the protein-synthesizing system does in DO infection. On the other hand, the not change the pattern of proteins synmessage for band B, present early in DO thesized in vitro directed by late T4 mRNA infection, is absent late in DO infection. (Fig. 10). The abundance distribution in Therefore it becomes necessary to divide the two cases is strikingly similar, including early messages into two subclasses with respect to their presence or absence late the large band at 35,000 mol. wt. whose origin is unknown. in DO infection. 2
4
6
8
206
WILHELM
AND
DISCUSSION
Our experiments demonstrate that a variety of structural proteins of T4, encompassing those of head, tail, and baseplate, may be synthesized in vitro with RNA from infected cells. A striking exception is the failure to synthesize the major proteins of the tail fibers, gene products 34 and 37, which are two of the largest phage proteins. Several possible explanations for this failure may be advanced. First, since the messages are necessarily large, they may be more susceptible to nuclease destruction during preparation than the smaller messages. This possibility is diicult to eliminate; however, the consistent failure of some twenty RNA preparations to make these proteins suggests something more basic. The genes involved in tail-fiber formation, genes 34-38, form a cluster which is displaced from the rest of the “late” region of the T4 genome. It is conceivable that the translation of the entire group requires some modification not present in the proteinsynthesizing systems from uninfected cells. Two observations make this explanation unlikely: (1) the product of one gene in the cluster, gene 38, of molecular weight approximately 25,000, can be made in such extracts in vitro; (2) the use of ribosomes or supernatant proteins from T4-infected cells in the system does not permit the synthesis of proteins 34 and 37 (J.W., unpublished observation). A third explanation, which we consider more likely, is based on the nature of the mRNA isolated. Morse et al. (1969) found that, upon de-repression of the tryptophan operon in E. coli, a wave of message synthesis occurs which is closely coupled to translation by a closely spaced cluster of ribosomes. This cluster is followed by message degradation from the 5’-end, which begins well before transcription of the operon has reached the 3’-end. Morse et al. calculated that the cluster of ribosomes would encompass a message length of some 20004000 nucleotides; this might be the length of message protected from nuclease action. The T4 gene products 37 and 34, of molecular weights 114,000 and 166,000, require
HASELKORN
messages of 3000-5000 nucleotides. Therefore, if the coupling of transcription, translation, and degradation in T4-infected cells is analogous to that observed with the tryptophan operon, functionally intact messengers for the uncoupled synthesis of these proteins in vitro simply may not be obtainable from infected cells. Our experiments answer some questions about the kind of RNA required for synthesis of the structural proteins. It is clear that RNA populations from cells early in infection are not effective; synthesis of these proteins is achieved only with RNA taken during the period when the infected cells are making the “late” RNA defined by hybridization-competition to experiments (Bolle et al., 1968a). In addition, in the course of infection of the nonpermissive host with DNA-negative mutants or with mutants in gene 55, little or no late messenger is ever made (Bolle et al., 196Sb). In agreement, we find that populations of RNA made late in DO or gene 55 infection do not direct the synthesis of the late, structural proteins. We note further that the in vitro synthesis of T4-specific proteins occurs in cell-free extracts derived from uninfected cells. This observation, plus the correlation of late message with the synthesis of late proteins, lends further support to the concept that the control of T4 development is largely transcriptional, and that translational modifications play no major, positive, roles. The transcription of early messages in T4 infection is under both positive and negative control. When cells are infected in the presence of chloramphenicol, only immediate early species of RNA are made. Delayed early species require the expression of one or more immediate early genes for their transcription (Salser et al., 1970; Milanesi et al., 1970). The internal proteins are made in vitro by chloramphenicol RNA (L. RI. Black, Jr. and L. Gold, personal communication). We find only a few one correspondmg in bands, including position to the largest internal protein, directed by chloramphenicol RNA (J. M. W., unpublished). The majority of bands whose synthesis is directed by early RNA
IN
VITRO
SYNTHESIS
OF T4 PROTEINS
are delayed early proteins. Among these, there appear to be three modes of negative control. The shut off of one class requires DNA replication and gene 55 function. Another class is shut off without gene 55, and a third, represented by band B in Fig. 9, is shut off even in the absence of DNA replication. ACKNOWLEDGMENTS This research was supported by a grant from the National Institutes of Health (AI-GM-09315). One of us (J.M.W.) was the recipient of a postdoctoral fellowship from the American Cancer Society. We t.hank 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. (1968a). Transcription during bacteriophage T4 development: Synthesis and relative stability of early and late RNA. J. Mol. Biol. 31, 325-348. BOLLE, A., EPSTEIN, R. H., S~LS~R, W., and GEIDUSCHEK, E. P. (1968b). Transcription during bacteriophage T4 development : Requirements for late messenger synthesis. J. Mol. Biol. 33, 339362. COOLSM~, J., and HASELKORN, R. (1969). In vitro synthesis of T4 proteins. Biochem. Biophys. Res. Commun 34, 253-259. EDGAR, R. S., and LIEL~USIS, I. (1965). Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52, 1187-1200. EPSTEIN, R. H., BOLLE, A., STEINRF:RO, C. M., KELLENBERGER, E., BOY DF, L.* TOUR, E., CH~:VALLEY, R., EDGAR, R. S., SUSM~N, M., DENHARDT, G. H., and LIsLaUSIS, I. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. FAIRBANKS, G., JR., LEVINTHAL, C., and REF,DER, 1~. H. (1965). Analysis of X-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393-399. FLATGA.~RD, J. (1969). The role of the gene 9 product in the assembly and triggering of bacteriophage T4. Thesis. California Institute of Technology. GOLD, L. M., and SCHWEIGER, M. (1969). Synthesis of phage-specific a- and @glucosyl transferases directed by T-even DNA in vitro. Proc. Nat. Acacl. Sci. U.S. 62,892X398.
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HOSOD~, J., and LEVINTHAL, C. (1968). Protein synthesis by E. coli infected with bact,eriophage T4D. Virology 34,709-727. Hsu, W. T., and WEISS, S. B. (1969). Selective translation of T4 template RNA by ribosomes from T4-infected E. coli. Proc. Nat. Acad. Sci. u. s. 64,345-351. KING, J. (1968). Assembly of the t,ail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J. (1970). In vilro assembly of t.he bacteriophage T4 tail core: three sequential steps. Fed. Eur. Biochem. Sot. Symp. 21, 156. KING, J., and WOOD, W. B. (1969). Assembly of bacteriophage T4 t,ail fibers: the sequence of gene product interaction. J. fifol. Biol. 39, 503601. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. iVuture (London) 227, 680685. LAEMMLI, U. K., MOLBI~:RT, E., SHOUX, M., and KELLENBERGEH, E. (1970). Form-determining fun&ion of the genes required for the assembly of the head of bacteriophage T4. J. Mol. Biol. 49,99-114. MILANESI, G., BRODY, E. N., GRSU, O., and GEIDUSCHEK, E. P. (1970). Transcription of the bacteriophage T4 template in vitro: Separation of “Delayed Early” from “Immediate Early” transcript.ion. Proc. i\rat. Acod. Sci. U. S. 66, 181-188. MORSE, D. E. MOSTF:LLER, R. D., and Y.INOFSICY, C. (1969). Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spring Harbor Symp. Quant. Biol. 34, 725-740. R. (1970). SALSF,R, W., BOLLE, A., and EPSTEIN, Transcription during bacteriophage T4 development : A demonstration that distinct subclasses of “early” RNA appear at different times and that some are “turned off” at late t,imes. J. Mol. Biol. 49, 271-296. SARIBHAI, A. S., STRETTON, A. 0. W., BRENNER, S., and BOLLE, A. (1964). Co-linearity of the gene with the polypeptide chain. Nature (London) 201, 13-17. SCHW&XGER, M., and GOLD, L. M. (1969). Bact)eriophage T4 DNA-dependent in vitro synthesis of lysozyme. Proc. Nat. Acad. Sci. U. S. 63, 1351-1358. SMITH, F. L., and HAS~LKORN, IX.. (1969). Proteins associated with ribosomes in T4-infected E. coli Cold Spring Harbor Symp. Quant. Biol. 34, 91-94. WIBF,RG, J. S., DIRKSI”N, M., EPSTEIN, R. H., LURID, S. E., and &JCHANAN, J. M. (1962). Early enzyme synthesis and its control in E.
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coli infected with some amber mutants of bacteriophage T4. Proc. Nat. Acad. Sci. U. S. 48,293-302. 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. Quant. BioZ. 34, 793-798.
HASELKORN YOUNG, E. T. (1970). Cell-free synthesis of bacteriophage T4 glucosyl transferase. J. Mol. Biol. 51, 591-604. YOUNG, E. T., II, and VAN HOTJWE, G. (1970). Control of synthesis of glucosyl transferase and lysozyme messengers after T4 infection. J. Mol. BioZ. 51, 605-620.