stocks, Drs. Don Groth, Jack Kinkade, June R. Scott, Ray Shapira, and. Bob Fritz and Mr. Vaughn Kubiak for critical reading of the manu- script, and Mrs. Lee ...
Proc. NatI. Acad. Sci. USA Vol. 75, No. 5, pp. 2175-2179, May 1978
Biochemistry
Adenovirus DNA-directed transcription of 5.5S RNA in vitro (cell-free system/KB RNA polymerase III)
GUANG-JER WU Departments of Microbiology and Chemistry and The Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322
Communicated by Norman H. Giles, February 13,1978
A cell-free system developed from human KB ABSTRACT cells was used to transcribe 5.5S RNA from deproteinized adenovirus DNA in vitro. The cell-free RNA synthesis is dependent upon exogenous templates, ribonucleoside triphosphates, and cell-free postmitochondrial supernatant of human KB cells. The synthesis of 5.5S RNA is inhibited only by high levels of aamanitin; therefore it is carried out by RNA polymerase m. The rate of synthesis was linear for at least 2 hr, indicating reinitiation. The 5.5S RNA synthesized in vitro is similar to the corresponding in vivo RNA in size, sequence, and coding region on adenovirus type 2 DNA. In this report is demonstrated in vitro synthesis of a facsimile of an in vivo transcript directed by deproteinized DNA in a mammalian cell-free postmitochondrial supernatant system.
Animal viruses provide excellent model systems for probing the molecular control mechanisms of gene expression in eukaryotic cells. Studies on the molecular biology of adenovirus (Ad) have provided considerable information regarding expression of the viral genome, in particular, at the levels of transcription and post-transcriptional processing (1-8). During productive infection of Ad in human KB cells or HeLa cells, a single species of 5.5S RNA is produced in abundance at the late stage of infection. This RNA, which was also designated as virus-associated RNA, is 156 nucleotides long (4-6). The RNA is found predominantly in the cytoplasm. Its function is unknown. Recently, 5.5S RNA was shown to associate with a novel minor species of 5.2S RNA. The 5.5S RNA (virus-associated RNA1) and 5.2S RNA (virus-associated RNA2) hybridize to distinct but contiguous regions of the adenovirus type 2 (Ad 2) DNA; these are 28.5-30% and 30-82%, respectively, of the genome from the left-hand end (7, 8). Several groups have demonstrated that virus-associated RNA1 is initiated and synthesized in vitro from endogenous template in the nuclei isolated from late infected KB cells by an enzyme that resembles RNA polymerase III in its sensitivity to high levels of a-amanitin (9-11). However, the system of isolated nuclei is limited in use for studying the molecular mechanisms of transcription and regulation in vitro, since it is difficult to add or withdraw protein factors that cross the nuclear membrane (12, 13). The purpose of this study was to develop a mammalian cell-free and membrane-free system to investigate the molecular mechanism of synthesis and regulation of 5.5S RNA.
essential medium (F-13, GIBCO) supplemented with 5% horse serum (GIBCO) at 37'. For virus-infected cells, the KB cells were infected at a multiplicity of 100 plaque-forming units per cell. Preparation of Purified Adenovirus. The stocks of Ad types 2 and 5 (Ad 2 and Ad 5) were originally obtained from H. Ginsberg and H. Young, respectively. Both serological types were propagated according to Bello and Ginsberg (14). The infectivities of the virus stocks were quantified by plaque assay (15). Virus was purified by the procedure of Lawrence and Ginsberg (16). For isolating [3H]thymidine-labeled virus, the infected cells were labeled twice with [3H]thymidine (1.7 ,uCi/ml, Schwarz/Mann, 66 Ci/mmol) at 2 and 18 hr after infection and purified by the same procedure. Preparation of DNA. Both Ad 2 and Ad 5 DNA were prepared from the purified virus as described by Pettersson and Sambrook (17). DNA from the uninfected and infected KB cells was prepared from the isolated nuclei (12) by Marmur's procedure (18). Preparation of Cell-Free Extract for Transcription. KB cells were washed and homogenized as described (12). The supernatant fraction (S-20) was obtained from the homogenate by centrifugation at 20,000 X g for 10 min at 4° in a SS-34 rotor in a Sorvall centrifuge and used for DNA-dependent cell-free RNA synthesis. Conditions for Cell-Free RNA Synthesis. The reaction mixture (0.1 ml) contained 45 mM Tris-HCI (pH 7.9), 7 mM 2-mercaptoethanol, 1 mM dithiothreitol, 1 mM ATP, 0.25 mM GTP, 0.25 mM CTP, 25 ,uM UTP, 1JCi of [3H]UTP (Amersham/Searle, 55 Ci/mmol), 10.3 mM potassium phosphoenolpyruvate (pH 7.5), 40,uM each of the 20 L-amino acids, 4.5 mM MgCl2, 36 mM KCI, 30 ,l of S-20 (10 mg of protein per ml), and 1-2 ug of DNA. The reaction was carried out at 29° for 90 min unless specified otherwise and stopped by pipetting the mixture onto a piece of Whatman no. 3 paper which was washed and processed; radioactivity was then determined
(19). Preparation of RNA for Gel Electrophoresis and Hybridization. RNA was extracted from a 0.5-ml reaction mixture, containing all the necessary ingredients except that 250 uCi of [3H]UTP was used, by the method of Holmes and Bonner with slight modifications (13), and treated with RNase-free DNase
(20 Ag/ml) (19).
Unlabeled RNA was prepared from the cytoplasmic fraction of the cell homogenate (12). Poly(A)-containing RNA was removed from cytoplasmic RNA by retention on a cellulose column (Whatman CC41) (20). RNA (4-6S) was purified twice from the cytoplasmic RNA that did not contain poly(A) by sedimentation through a sucrose gradient (19). The purity of
MATERIALS AND METHODS Cells. Human KB cells were obtained from Flow Laboratories, Rockville, MD, and maintained as a suspension culture in exponential phase of growth in Joklik-modified minimum The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: Ad 2, adenovirus type 2; Ad 5, adenovirus type 5; KB(L) DNA, DNA isolated from a KB cell suspension culture infected with 100 plaque-forming units of Ad 2 per cell for 18 hr. 2175
2176
Biochemistry: Wu
RNA was tested by electrophoresis in 8% polyacrylamide gel. Gel Electrophoresis and Fluorography. RNA was separated by electrophoresis in 2% acrylamide/0.5% agarose composite slab gel (0.24 X 14 X 10.5 cm) and 8% acrylamide slab gel (0.12 X 14 X 10.5 cm) according to Studier (21) with minor modifications (13). All RNA samples were heated at 1000 for 30 sec before electrophoresis to eliminate aggregation. Both kinds of gel were processed and fluorographed (22). RNA-DNA Hybridization. DNA was fixed on 22-mm cellulose nitrate filters (Schleicher and Schuell, BA85) as described (23). Filter hybridization was done in 50% formamide for 72 hr at 370 and processed (13). Restriction Endonuclease Cleavage of Ad 2 DNA. The restriction endonucleases EcoRI and HindIII were purchased from Biolabs, Beverly, MA. Ad 2 DNA was cleaved with EcoRI (24) and with HindIII (25). The digested DNA fragments were purified (26), separated by electrophoresis in 1.4% agarose slab gel (27), and visualized with a UV lamp after they were stained by ethidium bromide. Blotting and Hybridization. Denatured DNA fragments separated in agarose gel were transferred to a cellulose nitrate strip (HAWP-304F0, Millipore) in a stream 1.5 M NaCl/0.15 M Na3 citrate, fixed, used for hybridization, and fluorographed at -700 as described by Southern (28). RESULTS Transcription Directed by Exogenous DNA in a Cell-Free System. Since small RNA is transcribed by RNA polymerase III (1i), a cell-free system was developed from the postnuclear fraction which contains most of the activity of RNA polymerase III in human KB cells. The S-20 fraction from uninfected KB cells or from KB cells 6 or 18 hr after infection by Ad 2 was equally effective in stimulating RNA synthesis directed by exogenous DNA. Therefore the S-20 fraction from uninfected KB cells was used for the cell-free synthesis, since no viral specific 5.5S RNA was present in this extract. In this system RNA synthesis is dependent on exogenous templates (Table 1). The optimal concentrations of MgCl2 and KCl are 4.5 mM and 36 mM, respectively. a-Amanitin was used to determine which one of the three major classes of RNA polymerases was responsible for RNA synthesis in this system. As indicated in Table 1, most of the RNA synthesis stimulated by Ad 2 DNA was due to the activity of RNA polymerase III, as determined by its sensitivity to high concentrations of a-amanitin (higher than 100 jg/ml). Ten percent of the RNA synthesis was inhibited by low concentrations of a-amanitin (0.1-0.5 jg/ml), suggesting some activity of RNA polymerase II in the system. Twenty percent of the RNA synthesis was due to the endogenous activity, which was able to incorporate [3H]UMP into cold trichloroacetic acidinsoluble radioactive material in the absence of exogenous template, that was resistant to a-amanitin (200 ,g/ml). The amount of RNA synthesized endogenously was not significantly different from that synthesized in the complete system without addition of three nucleoside triphosphates. Thus, the endogenous RNA synthesis was not due to the activity of RNA polymerase I. DNA isolated from KB cells (KB DNA), or KB cells infected by Ad 2 for 18 hr [KB(L) DNA] also stimulated RNA synthesis in the system. Most of the RNA synthesis stimulated by KB or KB(L) DNA was also due to RNA polymerase III, since it was sensitive to high but not low concentrations of a-amanitin. Kinetics of In Vitro Transcription Directed by Ad 2 DNA. The kinetics of in vitro transcription directed by Ad 2 DNA in
Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Characteristics of RNA synthesis in vitro Addition or omission
Activity,
Complete* -S-20 -Ad 2 DNA -Ad 2 DNA + a-amanitin (200 ,g/ml) -ATP, GTP, CTP + a-Amanitin (0.51Ag/ml) +a-Amanitin (200 ,g/ml) +Pancreatic DNase (200 jtg/ml) +Pancreatic RNase (20 ;ig/ml) +KB DNAt +KB DNAt + a-amanitin (0.5 ug/ml) +KB DNAt + a-amanitin (200 gg/ml) +KB(L) DNAt +KB(L) DNAt + a-amanitin (0.5 ng/ml) +KB(L) DNAt + a-amanitin (200 ,g/ml)
100 1 19 20 23 90 25 28 7 125 130 24 144 132 27
%
* The complete system (0.1 ml) contained Ad 2 DNA (1.3 jg/0.1 ml) and other ingredients as described in Materials and Methods. Incorporation of [3H]UMP into cold trichloroacetic acid-insoluble radioactive material in the complete system was 10096 (3864 cpm, specific activity of 1 pmol = 175 cpm). Incubation was for 90 min at 290. t KB DNA isolated from a KB cell suspension culture was used as the template (3.4 jig/0.1 ml) to replace Ad 2 DNA in the complete system. KB(L) DNA was used as the template (1.5 ,g/O.1 ml) to replace Ad 2 DNA in the complete system.
the cell-free system is shown in Fig. 1. The RNA synthesis stimulated by Ad 2 DNA was linear for at least 2 hr at 29°, except for a 10-min lag at the beginning of synthesis (as. shown in Fig. 1). At least 18 pmol of [3H]UMP is incorporated per 1.25 ,ug of Ad 2 DNA in a 90-min synthesis at 290. Because the pool size of UTP in the S-20 fraction was not determined, this is a minimal estimation. De Novo Synthesis of 5.5S RNA from Ad DNA In Vitro. The size of RNA synthesized in vitro directed by Ad 2 or Ad 5 DNA was analyzed by gel electrophoresis in two kinds of gel with different pore sizes. The fluorograms of the two slab gels are shown in Figs. 2 and 3. When Ad 2 or Ad 5 DNA was used as the exogenous template, an intensely labeled species of RNA appeared (slot f and h, Figs. 2 and 3). This species of RNA had the same mobility as the in vivo labeled 5.5S RNA (slot j, Figs. 2 and 3). The intensity of the 5.5S RNA synthesized in vitro in the fluorogram was not decreased by low concentrations of a-amanitin (0.1-0.5 Mg/ml) (data not shown), but was decreased by concentrations of a-amanitin higher than 100Mg/ml (slots g and i, Figs. 2 and 3). In order to show that the RNA synthesized in vitro was directed by exogenous templates and not by nonspecific stimulation of transcription of endogenous template in the S-20, RNA synthesized in vitro directed by KB or KB(L) DNA was also
analyzed by gel electrophoresis. An intensely labeled population of RNA with an average size of 8 S appeared (slot b in Figs. 2 and 3) when KB DNA was used as the sole exogenous template.
When KB(L) DNA, which contained both KB DNA and Ad 2 DNA, was used as the template, both 8S and 5.5S RNA appeared (slot c, Figs. 2 and 3) and the synthesis of both RNAs was inhibited by a-amanitin (slot d, Figs. 2 and 3). Thus, 5.5S RNA was synthesized de novo from an exogenous template containing Ad genome. Sequence of In Vitro Synthesized 5.5S RNA Is Homologous to Ad DNA and In Vivo 5.5S RNA. To establish that the 5.5S RNA was transcribed from Ad 2 DNA, RNA-DNA hy-
Biochemistry: Wu
Proc. Natl. Acad. Sci. USA 75 (1978)
2177
a b c de f 9 h i i #-45 i-38 -28
28-
.. I-18
18-p
1 X
E
~.10
_
ACV
o
/
/
_
_- 4
4-
/
_-5.5
C
j f//~~~~~ i0.
0
/
60
120
Time, min
FIG. 1. Kinetics of Ad 2 DNA-directed RNA synthesis in the S-20 cell-free extract of KB cells. The reaction mixture (0.1 ml) contained all ingredients as described in Materials and Methods with Ad 2 DNA (13 .g/ml) (0 and +) or without DNA (0). Duplicates were done with the reaction mixtures containing Ad 2 DNA at each time point (0 and +). All the syntheses were at 290 for different lengths of times. Incorporation of [3H]UMP into cold trichloroacetic acid-insoluble radioactive material was determined. The difference in counts in the presence and absence of Ad 2 DNA at each time point was plotted as the dashed lines (M).
bridization was done. The RNA synthesized in vitro from various templates [Ad, 2, KB, or KB(L) DNA] was hybridized to Ad 2 or KB DNA under conditions of excess DNA. RNA synthesized in vitro from Ad 2 DNA hybridized only to Ad 2 DNA but not to KB DNA. RNA synthesized from KB DNA hybridized only to KB DNA. RNA synthesized from KB(L) DNA hybridized to both Ad 2 and KB DNA (data not shown). An RNA-DNA competition hybridization was carried out to compare the sequence of 5.5S RNA made in vitro to that made in vivo. Since only the 4-6S RNA from Ad 2-infected KB cells (18 hr after infection) contained the 5.5S RNA, the in vitro 5.5S RNA was competed preferentially by the 4-6S RNA isolated from the cytoplasmic RNA, that did not contain poly(A), of Ad 2-infected KB cells (18 hr after infection) (Fig. 4). In Vitro 5.5S RNA Is Transcribed from the Region of Ad 2 Genome Encoding for 5.5S RNA. The in vitro 5.5S RNA was hybridized to restriction endonuclease fragments of Ad 2 DNA which were transferred from gels to cellulose nitrate membrane by blotting (28). The EcoRI, HindIII, Hpa I, and BamHI restriction endonucleases were used to cleave Ad 2 DNA. The region encoding for the 5.5S RNA is contained in the EcoRI-A (0-58.5%), the HindIII-B (16.7-32.2%), the HpaI-A (28.557.4%), the BamHI-B (0-29.1%), and the BamHI-D (29.140.9%) fragments of Ad 2 DNA (7, 8). Therefore the RNA synthesized in vitro from Ad 2 DNA should only hybridize to these fragments. Unpublished results and those in Fig. 5 confirm the expectation. This locates the region on Ad 2 DNA from which the 5.5S RNA is transcribed in vitro to 28.5-32.2% of the genome from the left-hand end. This is the same region encoding for the 5.5S RNA synthesized in vivo (7, 8).
FIG. 2. Fluorograph of in vitro [3H]RNA in 2% acrylamide/0.5% agarose composite slab gel. (a) KB RNA labeled in vivo with [3HJuridine. KB cells (50 ml) in spinner culture were labeled with [3H]uridine (5 gCi/ml) for 24 hr. RNA was prepared from whole cells. (j) RNA labeled in vivo with [3H]uridine from KB cells infected with Ad 2. KB cells (50 ml) in spinner cultures were infected with 100 plaque-forming units per cell of Ad 2 for 15 hr and then labeled with [3H]uridine (10 ACi/ml) for 5 hr (15-20 hr after infection). [3H]RNA was synthesized in vitro, using different DNAs as template in the presence or absence of a-amanitin (200 gg/ml): (b) KB DNA, (c) KB(L) DNA, (d) KB(L) DNA + a-amanitin, (e) without DNA, (f) Ad 2 DNA, (g) Ad 2 DNA + a-amanitin,(h) Ad 5 DNA, and (i) Ad 5 DNA + a-amanitin. All in vitro RNAs were deproteinized and processed in the same way, as described in Materials and Methods, and dissolved in 100 Ml of H20; 10 ul of each was mixed with an equal volume of twice concentrated sample buffer, heated, and used for electrophoresis. The radioactivities of [3HJRNA used in electrophoresis were: (a) 102,000 cpm, (b) 22,000 cpm, (c) 33,000 cpm, (d) 23,000 cpm, (e) 3000 cpm, (f) 16,000 cpm, (g) 4500 cpm, (h) 11,000 cpm, (i) 4000 cpm, and (j) 39,000 cpm. The processed gel was fluorographed at -70° for 3 days.
DISCUSSION The Ad genome codes for a single 5.5S RNA species which is actively transcribed by RNA polymerase III in Ad 2-infected KB cells. This may serve as an excellent model system for cellfree synthesis of eukaryotic RNA in a DNA-directed cell-free system for studying the molecular mechanisms of regulation of expression of eukaryotic genes. The cytoplasmic fraction prepared from various cell lines (human HeLa, KB and Adinfected KB cells, Chinese hamster ovarian cells, and murine a
5.5-
b c d e
g
h 'ii
---to
4-U FIG. 3. Fluorograph of in vitro [3HJRNA in 8% acrylamide slab gel. All the RNA samples used were the same as described in Fig. 2. The radioactivities of [3H]RNA used for electrophoresis were: (a) 67,000 cpm, (b) 14,000 cpm, (c) 22,000 cpm, (d) 15,000 cpm, (e) 2000 cpm, (f) 16,000 cpm, (g) 4500 cpm, (h) 7000 cpm, (i) 3000 cpm, and Ci) 90,000 cpm. The processed gel was fluorographed at -70° for 7 days.
Biochemistry: Wu
2178
Proc. Natl. Acad. Sci. USA 75 (1978)
If
.HindlI
.EloRIl. A--
:2
*N
_
B-
.0
r
C
DE D___-
z a:
_-
H--
Competing RNA, Mg/ml
FIG. 4. Hybridization of in vitro [3H]RNA to Ad 2 DNA in the presence of competing 4-6S RNA, prepared from KB or Ad 2-infected KB cell cytoplasm. In vitro [3HJRNA synthesized from Ad 2 DNA was purified once by sedimentation in a sucrose gradient and the fractions of the 4-6S region were pooled for hybridization. Hybridization was done in 1 ml of hybridization solution (Materials and Methods) containing 1900 cpm of pooled in vitro [3H]RNA and a blank filter and a filter containing 30 fg of Ad 2 DNA in the presence of different concentrations of competing 4-6S RNA. The competing 4-6S RNA was prepared from the nonpoly(A)-containing cytoplasmic RNA of KB cells (0) or Ad 2-infected KB cells (18 hr after infection) (@) and purified. The counts on blank filters were subtracted from those on the DNA-containing filters. [3H]RNA counts (950 cpm) retained on the DNA-containing filters after hybridization in the absence of competing RNA were normalized to 100%.
Krebs II ascites tumor cells) stimulates isolated nuclei to synthesize small RNA (4-8S) (refs. 12, 13, and unpublished data). This suggests that there are factors present in the cytoplasmic fraction responsible for this stimulation. Roeder's group (29) has demonstrated that 90% of the cellular RNA polymerase III activity was found in the cytoplasmic fraction when aqueous buffer was used for preparing the cell homogenate from mouse myeloma cells. Thus, it was logical to prepare a cell-free system from the postnuclear fraction supplying RNA polymerase III for the transcription of small RNA from eukaryotic DNA. Evidence is presented in this report that the 5.5S RNA corresponding to that made in vivo from Ad 2 DNA can be synthesized de novo (and possibly subsequently processed) from the deproteinized native Ad DNA by RNA polymerase III in a crude cell-free postmitochondrial supernatant derived from human KB cells. The synthesis of 5.5S RNA in vitro is dependent on Ad 2 DNA and three ribonucleoside triphosphates, and is sensitive to a-amanitin. The 5.5S RNA synthesized in vitro hybridizes only to Ad 2 DNA but not to KB DNA. It is therefore unlikely that the synthesis of 5.5S RNA in vitro is merely due to the transfer of [3H]poly(U) to KB cellular 5S rRNA. The kinetics of synthesis is linear for 2 hr, suggesting continued initiation of RNA synthesis in vitro. Since most of the DNA-dependent RNA synthesis from Ad 2 DNA is due to transcription of 5.5S RNA, a minimal estimate of 10 molecules of 5.5S RNA is initiated per molecule of Ad 2 DNA in a 90-min incubation at 290. The 5.5S RNA made in vitro hybridizes to the region on the Ad 2 genome encoding for 5.5S RNA gene. The studies of RNA-DNA competition hybridization suggest that the sequence of the in vitro 5.5S RNA is homologous to that of the in vivo 5.5S RNA. This is further supported by the results of fingerprint analyses of T1 RNase digests of 5.5S RNA made in vivo and in vitro (unpublished data). The evidence presented suggests that initiation and termination sequences on native viral DNA are correctly recognized in vitro by RNA poly-
J-
KFIG. 5. Hybridization of in vitro [3HJRNA to EcoRI and HindIII restriction endonuclease-cleaved fragments of Ad 2 DNA. Ad 2 DNA (6 ,ug in 30 ul) was digested with 30 units of EcomI or 36 units of HindIII at 370 for 60 min, purified, separated by electrophoresis in 1.4% agarose slab gel, stained by ethidium bromide, denatured,
transferred by the blotting method of Southern, fixed, and used for hybridization. [3H]RNA (38,000 cpm) synthesized from Ad 2 DNA in vitro was used for hybridization to the cellulose nitrate strip containing either EcoRI fragments (a) or HindI11 fragments (c) of Ad 2 DNA. After hybridization, the strips were dipped in toluene solution containing 20% 2,5-diphenyloxazole, and fluorographed at -70° for 7 days. For markers, Ad 2 [3H]DNA prepared from [3Hjthymidinelabeled virus by the method of Pettersson and Sambrook (17) (24,000 cpm in 15 1l) was digested with 20 units of EcoRI restriction endonuclease (b) or 24 units of HindIII restriction endonuclease (d), purified, separated on gel, denatured, transferred, fixed on cellulose nitrate sheet, dipped in a toluene solution containing 20% 2,5-diphenyloxazole, and fluorographed at -70° for 7 days.
merase III in this system. Although there is no evidence concerning the existence of a precursor for 5.5S RNA, the high efficiency of production of apparently fully mature 5.5S RNA in vitro suggests that either there is no precursor or that there is tight coupling of transcription and post-transcriptional processing. This requires further investigation. Since 5.5S RNA is synthesized from Ad DNA by the S-20 fraction prepared from uninfected, early infected, or late infected KB cells, it seems likely that only host enzymes are required for the transcription of the 5.5S RNA gene. The presence of 5.5S RNA in abundance at the late stage of infection may be due to the presence of large quantities of Ad DNA at the late stage of infection and to the high rate of transcription by RNA polymerase III from the promoter of 5.5S RNA gene. In addition to the 5.5S RNA (156 nucleotides long), there are six species of small RNA, 190, 130, 120, 110, 105, and 100 nucleotides long, transcribed in vitro in this system by RNA polymerase III (slots c, f, and h in Fig. 3). The synthesis of these RNAs is inhibited only by # high concentration of a-amanitin (200 ug/ml) (slots d, g, and i in Fig. 3). Since all these six RNA species are also synthesized in vivo (slot j in Fig. 3), it is unlikely that they are artifacts of the in vitro system. There is a population of RNA with a molecular weight of 1 X 105 (about 8 S) synthesized from KB DNA by RNA polymerase III (Figs. 2 and 3). An RNA of similar size was also syntheiszed in abundance when the isolated nuclei system was
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Wu used (13, 30). Preliminary results of competition hybridization indicate that there is no sequence homology between the in vitro 8S RNA and the in vivo KB 4-6S RNA (data not shown). This RNA has not been further characterized. In this system there is a small quantity of 5S RNA synthesized from KB DNA. This suggests that RNA polymerase III is able to recognize the correct promoters on both viral DNA and KB DNA. Other groups have demonstrated that no mature 5S rRNA is synthesized by purified RNA polymerase III when the isolated DNA containing 5S rRNA gene is used as the template (31) unless chromatin is used as the template (32, 33) or the DNA is injected into frog oocytes (34). This report suggests that a mature-sized 5S RNA and 5.5S RNA can be synthesized from deproteinized native DNA when a crude enzyme system is used for in vitro transcription. Recent work by Roeder's group (35) has shown that a correctly sized 5.5S RNA could not be made in vitro by highly purified KB cell RNA polymerase III using deproteinized Ad 2 DNA as the template. This further supports the possibility that factors other than the KB RNA polymerase III are required for faithful transcription of Ad 2 DNA. Since this study has clearly demonstrated that the in vitro system mimics the in vivo situation, it is possible to study further whether factors other than RNA polymerase III are required for correct transcription and how RNA polymerase III and such factors interact with the promoter site on the 5.5S RNA gene. I thank Drs. H. Ginsberg and H. Young for supplying adenovirus stocks, Drs. Don Groth, Jack Kinkade, June R. Scott, Ray Shapira, and Bob Fritz and Mr. Vaughn Kubiak for critical reading of the manuscript, and Mrs. Lee Thorn, Mrs. Joel Driscoll, and Peggy Tyler for typing the paper. This work was supported by Cancer Center Core Support Grant CA16255 from the National Cancer Institute and partially supported by Biomedical Research Support Grant RR5364 from the National Institutes of Health.
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