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A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis
J. Mol. Biol. (1974) 84, 173-183
A Tentative Initiation Inhibitor of Chromosomal Heterogeneous RNA Synthesis ENDREEQYHAZI Department of Histology Karolinska Institutet S-10401, Stockholm 60, Sweden (Received 9 July 1973, and in revised form 10 November 1973) The nucleoside analogue 5,6-dichloro-l-6-n-ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus ten&w but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRBt was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (7~5 100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When a-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulsechase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary glandcells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.
1. Introduction Several chemically distinct groups of antibiotics have been discovered which inhibit DNA-directed RNA synthesis. RNA is produced in reactions involving template DNA, polymerizing enzymes and nucleoside triphosphates, and RNA synthesis can, therefore, be interfered with at the level of each of these components. The best characterized inhibitor of RNA synthesis is actinomycin D, which binds to DNA and thereby disrupts its role as a template (Kersten et al., 1960; Goldberg et al., 1962; Harbers & Mtiller, 1962). Bacterial DNA-dependent RNA polymerases are effectively blocked by the antibiotic rifampicin (Wehrli et al., 1968; Jacob et al., 1970; Chambon et al., 1970). The best established inhibitor of a eukaryotic polymerase is the mushroom poison u-amanitin, which selectively inhibits the nucleoplasmic polymerase II, while the nucleoplasmic polymerase III and the nucleolar polymerase I are resistant towards inhibition by u-amanitin (Stirpe & Fiume, 1967; Lindell et al., 1970; Roeder & Rutter, 1970). Both u-amanitin and rifampicin exert their inhibitory effects by binding to the RNA polymerases. Furthermore, RNA synthesis can be t Abbreviations used: DRB, geneous nuclear RNA.
6,0-diohloro-l-B-n-ribof~anosylbenzimidazole;
173
HnRNA,
hetero-
174
E. EGYHAZI
suppressed by nucleoside analogues, which usually exert their inhibitory action by interfering with the intracellular nucleoside triphosphate pools. Both the template inhibitor, actinomycin D (Maitra et al., 1967; Hyman 8z Davidson, 1970), and the polymerase II inhibitor, a-amanitin (Seifart & Sekeris, 1969; Chambon et al., 1970; Jacob et al., 1970; Kedinger et al., 1970), interfere with the elongation of the growing RNA chain, although by different mechanisms. By contrast, rifampicin, the inhibitor of prokaryotic polymerases, acts on the initiation of RNA transcription but has no effect on the propagation of nascent RNA chains (Sippel &, Hartmann, 1968; Mosteller & Yanofsky, 1970). No inhibitor of any nuclear RNA synthesis has been described that acts at the level of initiation. The effect of the purine nucleoside analogue, 5,6-dichloro-I-/3-n-ribofuranosylbenzimidazole, on RNA synthesis in explanted salivary glands of Chironomus tentans has been tested earlier (Egyhazi et al., 1970; Edstrom et al., 1971). These analyses showed that DRBt selectively inhibits synthesis of chromosomal heterogeneous high molecular weight RNA. In similar experiments the effect of a-amanitin was investigated (Egyh4zi et a+?.,1972). Gel electrophoretic data revealed that the polymerase inhibitor a-amanitin, like the nucleoside analogue DRB, selectively abolishes the labelling of chromosomal HiiRNA. While a-amanitin inhibits the elongation of growing RNA chains the mechanism of inhibition by DRB is unknown. It was therefore decided to study further the inhibitory effect of DRB on synthesis of HnRNA. a-Amanitin and actinomycin D were used for comparison due to their well established effects on RNA chain propagation. The data reported here show that DRB suppresses labelling of chromosomal HnRNA by a mechanism different from that of a-amanitin and actinomycin D. This finding, in conjunction with the analogy between DRB and rifampicin as expressed in the sequence of inhibition, suggests that the nucleoside analogue inhibits HnRNA synthesis at the level of initiation.
2. Materials and Methods (a) Biological material and labelling conditions Late-fourth larval instar animals of the species Chironcrmus tentans were used, cultured as described by Beermann (1952). In each experiment 4 animals were used. Four salivary glands were explanted into 50 ~1 of modifled Cannon’s medium (Cannon, 1964; Ringborg & Rydlander, 1971) supplied with 100 &?i of [3H]cytidine and 100 PCi of [3H]uridine (28 Ci/mmol and 25 Ci/mmol, respectively) and with actinomycin D (10 pg/ml), a-amanitin (40 pg/ml), or DRB (20 pg/ml). The sister glands were incubated in a similar way but without the drugs (control glan&). As a rule, incubations were carried out for 45 and 90 mm at 18°C. Actinomycin D and DRB were obtained from Merck, Sharp and Dohme while a-amanitin wig a gift from Dr T. Wieland. (b) Isolation of chromoeomea After incubation, the glands were fixed in ethanol/acetic acid (3 : 1) at 4°C for 30 mm and rinsed 3 times for 10 min each in 70% ethanol at 4’C, and finally transferred to a mixture of ethanol/glycerol (1: 1) for 60 min at 4°C. The glands were then dissected under liquid parafikr, according to Edsttim (1964). Chromosomes I to III from 24 cells (4 glands) were pooled. (c) Extra&ion of RNA The pooled chromosomal sample was digested for 5 to 10 min in hanging drops in the liquid paraffin environment with a preincubated solution of O-02 M-Tris*HCl buffer, pH t SW footnote
on p. 173.
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RNA
176
7.4, containing sodium dodecyl sulphate (5 mg/m.l) and nuclease-free pronase (1 mg/ml) (Calbiochem). The dissolved sample was then transferred by means of a small piece of filter paper to 20 (~1 of E buffer (0.02 M-Tris~HCl buffer, pH 8.0, 0.02 M-NaCl, 0.002 MEDTA and 2 mg sodium dodecyl sulphate/ml), and 30 ag of Eschetichia coli RNA was added as marker (Egyhazi et al., 1969). As a rule, electrophoresis was performed immediately after extraction. (d) Electrophoresis
and rneasuremeti
of radioactivity
The RNA digest was subjected to electrophoresis in 1 y0 agarose gels. The gel slabs were prepared in E buffer and the electrophoresis was carried out as described by Daneholt (1973). At the end of the run, the gel was sliced and the slices were then transferred to Packard scintillation vials, each containing 10 ml of toluene scintillator (which in 1 1
contains
30 ml soluene (Packard),
ethanol), incubated overnight spectrometer at an efficiency
5.5 g of Permablend
at 37°C and &rally of around 33%.
counted
(Packard) in a Packard
and 20 ml methoxyliquid-scintillation
3. Results (a) Labelling
of chromosomd
RNA
in the absence of inhibitors
The electrophoretic analyses of labelled RNA from chromosomes I to III after 45 and 90 minutes of incubation with tritiated nucleosides are presented in Figure 1.
The radioactivity profile of 45-minute labelled chromosomes shows a bimodal distribution; there is a peak in the 4-8 S range and label with a variable, heterogeneous distribution
in the E-100
S range as reported
earlier
(Daneholt
et al., 1969). When
4s
I 2c
I 30
I 40
Slice no.
FIG. 1. Electrophoretic separations of chromosomal RNA after 46 and 90 min of labelling. Four glands from 4 different animals were incubated in 60 al of incubation medium oontaining 100 8Ci each of cytidine and of uridine, and their sister glands were plaoed in another 60 81 of the same medium. The glands were incubated at 18’C for 46 min and 90 min, respectively. After incubation the glands were fixed and from each gland 6 sets of chromosomes I to III were isolated by dissection. The labelled RNA from each sample was released by pronase-sodium dodecyl snlphate treatment, electrophoresis was carried out in 1 oh agarose gel and the radioactivity wes counted in a Packard liquid-scintillation spectrometer. E. coli RNA was used as marker (23 S, 16 S and 4 S). The positions of 76 S and 30 S were determined in parallel analyses of Balbiani ring 2 RNA and nucleolar RNA, respectively. For more details see Materials and Methods. -a-e--, 46-Min labelling; -O-O-, 90-min labelling.
176
E. EGYHAZI
the time of incubation was prolonged to 90 minutes the migration pattern of chromosomal RNA showed a parallel increase of radioactivity over the entire size spectrum. The RNA of chromosome IV, dominated by Balbiani ring 2 RNA, possesses labelling kinetics different from those of chromosomes I to IH, and, therefore, the present analyses of chromosomal RNA will not include RNA from chromosome IV. The finding that HnRNA has not acquired maximum labelling after 45 minutes, and that there is a further increase in the radioactivity pattern between 45 and 90 minutes, may have different reasons : (1) a slow saturation of chromosomal nucleotide pools with labelled nucleosides; (2) accumulation of finished HnRNA on the chromosomes ; (3) the time required for the completion of larger HnRNA molecules is longer than 45 minutes. This latter alternative is unlikely because it would imply a shift in the pattern after 90 minutes labelling towards the high molecular weight range in comparison to that after 45 minutes labelling. More extensive data are required to decide this question finally. (b) Inhibition
of heterogeneous nuclear RNA
by DRB
(i) Chase type of experiments Our previous work demonstrated that DRB reduced labelling of chromosomal HnRNA, at least in the 30-100 S range, to 5 to loo/, of the normal values (Egyhazi et al., 1970). Labelling of chromosomal4-8 S RNA and nucleolar preribosomal RNA was only moderately affected. The labelling of the glands in these studies was, however, carried out after 60 minutes of preincubation in the presence of the nucleoside analogue. While this approach was useful in establishing the gross effect on synthesis of nuclear RNA of various types, more appropriate experimental conditions are achieved by the chase type of experiments, which can provide information on the sequence of inhibition of chromosomal HnRNA synthesis. The presence of DRB in the incubation medium for 45 minutes, after 45 minutes of labelling in the absence of the drug, resulted in a differential change in the radioactivity pattern of chromosomal HnRNA compared to the profile of normal RNA after 45 minutes of labelling (Fig. 2(a)). Incorporation in the 4-8 S range seems not to be affected by DRB in accordance with previous observations (Egyhazi et al., 1970). Whereas there is, during DRB treatment, a continuous RNA synthesis in the high molecular weight range (75-100 S), the label associated with the smaller size range of HnRNA (below 75 S) is reduced. The degree of decrease of radioactivity in HnRNA seems to be inversely related to the size of RNA molecules. When the chase period with DRB is prolonged to 90 minutes the residual HnRNA labelling is terminated and a considerable drop in label can be recorded also in the higher molecular weight range of the spectrum (Fig. 2(b)). By contrast, the nucleoside analogue-insensitive radioactivity in the 16-30 S region shows an increase. (ii) Residual synthesis of RNA derived from chromosomespreincubded with nudeaside analogue The data described in Figure 2 reveal that a time interval of 45 to 90 minutes is required to interrupt labelling of 75-100 S RNA. On the other hand, the uptake of label by the low molecular weight HnRNA has already decreased after 25 minutes. To illuminate further the phenomenon of differential inhibition of HnRNA labelling by DRB, another line of approach was adopted. Salivary glands were preincubated
INITIATION
(75 S)
INHIBITOR
(30s)
OF CHROMOSOMAL
16s
OO Slice (a)
RNA
I IO
I 20
I 30
40
1
no
(bl
2. Eleotrophoretic separations of chromosomal RNA after labelling in absence of DRB and after chase with DRB. In each experiment 4 glands from 4 different animals were incubated in 50 ~1 of incubation medium containing 100 &i each of cytidine and of uridine, and their sister glands were placed as controls in another 60 4 of the same medium. The glands were incubated at 18°C for 46 min. While the incubation of control glands was interrupted after this time, the labelling of the other glands proceeded for an additional 46 min (a) and 90 min (b) after addition of DRB (20 pg/ml). Six sets of chromosomes I to III were dissected from each gland. For other data see the legend to Fig. 1. -@-a--, Labelling in the absence of DRB ; -O-O-, labelling in the presence of DRB. FIG.
for a brief period in the presence of DRB before the labelled nucleosides were introduced into the incubation medium. In Figure 3 are shown the results of a Xi-minute incubation with the drug preceding a labelling period of 30 minutes in the continued presence of the drug. The control glands were subjected to the same type of treatment, but in the absence of inhibitor. A comparison between the electrophoretic profiles of drug-treated and normal chromosomes confirms the chase data, in that a residual labelling of HnRNA is still taking place, though at a decreasing rate. Furthermore, DRB affects labelling to a greater extent in the low molecular weight range (in the vicinity of 30 S) than in the range around 75 S. The 1630 S range constitutes an exception for three different reasons. (1) A relatively large peak, resistant to DRB but also to actinomycin D and RNAase, in the 1630 S range superimposes the label of HnRNA; (2) some radioactivity remains, even after prolonged pretreatment with DRB evenly distributed over the whole HnRNA spectrum; (3) accumulation of preribosomal RNA of nucleolar origin in the 1830 S range contributes to the pattern of HnRNA (Ringborg & Rydlander, 1971). If the time of preincubation was extended 12
178
E. EGYHAZI
Slice no
Fro. 3. Electrophoretic separations of chromosomal RNA labelled for 30 min in the absence and in the presence of DRB titer 15 min of preincubation. Four glands were incubated st 18’C for 16 min in 60 ~1 of incubation medium conteining DRB (20 rg/ml). They were then transferred to another 50 ~1 of the same medium contkning DRB, 100 @i each of cytidine and of uridine and incubated for 30 min at 18’C. For labbelling with tritieted nucleosides in the absence of DRB, the sister glands were used in an otherwise parallel procedure. Six sets of chromosomes I to III were dissected from each gland. For other details see the legend to Fig. 1. --@-a-, Normal cells; -c-O-, DRB-treated cells.
to 45 minutes in combination with a 45-minute labelling period, DRB inhibited labelling also in the high molecular weight range (Fig. 4). However, some radioactivity remains evenly distributed over the whole HnRNA spectrum due either to elevated background level or incomplete inhibition. (c) Control experiments (i) a-Amanitin
The ability of a-amanitin to impair the elongation of polymerase D-promoted transcription of RNA in vitro is well established (Chambon et al., 1970; Lindell et al., 1970; Roeder & Rutter, 1970). Further, it has been demonstrated that u-amanitin selectively inhibits labelling of chromosomal HnRNA in salivary gland cells of chironomids (Beermann, 1971; Serfling et al., 1972; Egyhazi et al., 1972), and as much as 5 to 10% of the normal values in the 30-100 S range in Chironomw tentans. The synthesis of 4-8 S RNA is not affected, and there is a-am&n&in-resistant label in the 16-30 S region. Thus or-amanitin is suitable as a reference inhibitor in studies with DRB. a-Amanitin in its capacity as an inhibitor of the propagation of growing RNA chains should stop, under chase conditions, the synthesis of all sensitive HnRNA fractions irrespective of the length of nascent polynucleotides. This expectation is
INITIATION
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RNA
179
Slice no.
Pm. 4. Electrophoretic separations of chromosomal RNA labelled for 46 min in the absence and in the presence of DRB after 45 min of preincubation. Four glands were incubated at 18°C for 46 min in 60 ~1 of incubation medium containing DRB (20 pg/ml). They were then transferred to another 60 ~1 of the same medium containing DRB, 100 pCi each of cytidine and of uridine and incubated for 46 min at 18°C. For labelling with tritiated nwleosides in the absence of DRB, the sister glands were used in an otherwise parallel procedure. For other details see the legend to Fig. 1. -m-e--, Norma1 cells; --O--O-, DRB-treated cells.
borne out by the results presented in Figure 5, where the electrophoretic pattern of RNA from u-amanitin-treated chromosomes is compared to that from normal 45minute labelled chromosomes. The radioactivity pattern of RNA from treated glands exhibits a generally decreased labelling in the range above 4-8 S. Incorporatjion of label in the 4-8 S region is not affected by cc-amanitin since these RNA fractions are insensitive to it. (ii) Actinomycin D Actinomycin D, known as an inhibitor of elongation of all DNA-dependent RNA synthesis (Maitra et al., 1967; Hymen & Davidson, 1970), depressed labelling of RNA in Chironomus tentans by up to 98%, at least in the 30-100 S range. Salivary glands incubated for 45 minutes with tritiated nucleosides in the absence of actinomycin D were incubated for another 45 minutes in the presence of the drug. Control glands were labelled for 45 minutes. The electrophoretic analyses display a synthesis inhibition by actinomycin D, with a concomitant decay of radioactivity in the whole HnRNA range above 30 S (not shown) as was also demonstrated by chase with uamanitin.
E. EGYHAZI
180
3
.0_111-7 Slice no.
FIG. 6. Eleotrophoretic separations of chromosomal RNA in the absence of a-amanitin and sfter a chase with cr-amanitin. In each experiment 4 glands from 4 different animals were incubated in 60 ~1 of incubation medium oontaining 100 PCi both of cytidine and of uridine, and their sister glands were placed as controls in another 60 ,ul of the same medium. The glands were incubated at 18°C for 46 min. While the incubation of control glends was interrupted after 46 min, the lebelling of other glands proceeded for another 46 min after addition of a-amanitin (40 pg/ml). Six sets of ahromosomes I to III were disseated from each gland. For other data see the legend to Fig. 1. -@-a--, L&belling in the absence of drug; -O--O-, l&belling in the presence of drug.
4. Discussion (a) Dichlororibofurctnosylbenzimidazole
preferentially inhibits the
synthesis of small heterogemus nuclear RNA
molecules
Administration of DRB after an initial l&belling period of 46 minutes diminished the radioactivity in the electrophoretic pattern starting in the lowest molecular weight region. It did, however, permit a continuous increase of l&belling in the 75-100 S range during a chase period of 45 minutes. The increased label in the 75-100 S range could be explained by the fact that HnRNA at the onset of DRB treatment had not attained maximum l&belling, as discussed above. A prolongation of the DRB chase from 45 to 90 minutes gave a substantial drop of label also in the high molecular weight range of the spectrum. This would be the expected effect if DRB preferentially interrupts the synthesis of small HnRNA molecules, and HnRNA molecules in the 75-100 S region escape inhibition during a 45-minute chase. The differential inhibition is not due to DRB-resistant HnRNA fractions with slow electrophoretic migration, since after prolong&ion of the chase or after a long enough preincubation period with DRB, labelling of all HnRNA fractions is equally abolished.
INITIATION
INHIBITOR
OF
CHROMOSOMAL
RNA
181
It appears obvious that the mechanism of inhibition of HnRNA synthesis by DRB is different from that of u-amanitin or actinomycin D. Use of these inhibitors of RNA chain propagation to depress labelling of HnRNA results only in a quantitative alteration in the radioactivity pattern. The synthesis inhibition with actinomycin D or a-amanitin is accompanied by a parallel decay of label in the entire size-range of HnRNA without any appreciable shift in the profiles from the high molecular weight range towards the region of lower S values. (b) Is DRB an RNA polymerase inhibitor in living cells? There are a number of findings which make DRB unlikely as a template inhibitor similar to, for example, actinomycin D or nogalamycin (Ellem & Rhode, 1970). The nucleoside analogue (1) allows a transient RNA labelling for a limited period of time; (2) preferentially depresses label associated with the low molecular weight range of the HnRNA pattern, but later on abolishes l&belling of molecules with higher S values also; and (3) lacks influence on labelling of 4-8 S RNA with chromosomal location, and on ribosomal RNA synthesis. For similar reasons it is unlikely that DRB should exert its inhibitory action by interfering with the intranuclear nucleotide pools, which would require extremely compartmentalized chromosomal precursor pools. Available data support the involvement of only one ribonucleotide precursor pool in the synthesis of nucleolar and nucleoplaamic RNA (Wu & Soeiro, 1971). In view of these considerations, the remaining alternative, that DRB acts as a polymerase inhibitor, is most likely. This question should be finally decided in a reconstituted system with purified RNA polymerases. In vivo the active form of DRB is, however, likely to be the triphosphate derivative as is the case with other nucleoside analogues, like cordycepin (Klenow, 1963) and tubercidin (Acs et al., 1964). (c) The interference of DRB with the initiation
of heterogeneousnuclear RNA synthesis
While a-amanitin interacts with the elongation of polymerase II-promoted RNA transcription, it is unlikely that DRB interrupts HnRNA synthesis at the level of elongation. The present electrophoretic data indeed would be inconsistent with such an interpretation. Therefore, it appears relevant to ask whether DRB is an inhibitor of chain initiation. Although no direct proof can be given at the present time, the accumulated data speak strongly for the hypothesis that DRB interferes with the initiation of HnRNA synthesis. The effect of DRB on HiiRNA synthesis resembles that of rifampicin on bacterial RNA transcription. The latter is a well established inhibitor of RNA chain! initiation by interaction with bacterial RNA polymerases, without preventing the completion of polynucleotides that are being synthesized (Pato & von Meyenburg, 1970; Doolittle & Pace, 1970). Thus the analogy in the inhibitory action between rifampicin and DRB may be summarized in two points. First, the administration of drugs shortly before incubation with labelled nucleosides is associated with a persistence of labelling at a decreased rate, followed by inhibition of further radioactivity rise. Second, formation of small RNA molecules is blocked earlier than that of large molecules. Thus the analogy in inhibitory action between DRB and the initiation inhibitor rifampicin supports the idea that DRB, like rifampicin, is an inhibitor of initiation. The molecular mechanism of inhibition by DRB is unknown. However, the finding that adenosine, but not gnanosine, is capable of blocking the inhibitory
182
E. EGYHAZI
effects of DRB (Tamm et aZ., 1960; Egyhazi, unpublished results), might in conjuction with present data justify the following working hypothesis: RNA polymerases initiating the synthesis of HnRNA are sensitive to the action of DRB by competition between DRB or a DRB metabolite and ATP in the formation of the initiation complex. Nucleoside analogue metabolite molecules bound in lieu of ATP molecules to the initiation sites then block the addition of a following nucleotide. By contrast, elongating polymerases, i.e. polymerases that have already passed the initiation step at the time of addition of DRB, would escape inhibition. The skilful technical assistance of Miss Agneta Askendal is gratefully acknowledged. I am also obliged to Miss Hannele Jansson for typing the manuscript, Mm Chana Szpiro for cultivation of larvae and Dr J. Hyde for revising the English text. This investigation was supported by the Swedish Cancer Society and Karolinska Institutet (Reservationsanslaget) . REFERENCES Acs, G., Reich, E. t Mori, M. (1964). PTOC. Nat. Acad. Sci., U.S.A. 52, 493-501. Beermann, W. (1952). Chromosoma, 5, 139-198. Beermann, W. (1971). Chromosoma, 34, 152-168. Cannon, G. B. (1964). Science, 146, 1063. Chambon, P., Gissinger, F., Mandel, J. L., Jr, Kedinger, C., Gniazdowski, M., BEMeihlac, M. (1970). Cold Spring Harbor Symp. Quant. Bill. 35, 693-707. Daneholt, B. (1972). Nature New Biol. 240, 229-232. Daneholt, B., Edstrom, J.-E., Egyhazi, E., Lambert, B. & Ringborg, U. (1969). Chromo8oma, 28, 399-417. Doolittle, W. F. & Pace, N. R. (1970). Nature (London), 228, 125-129. Edstrom, J.-E. (1964). In Methods in Cell Physiology (Prescott, D., ed.), vol. 1, pp. 417447, Academic Press, New York. Edstrom, J.-E., Egyhazi, E., Daneholt, B., Lambert, B. & Ringborg, U. (1971). Chromosoma, 35, 431-442. Egyh&zi, E., Daneholt, B., Edstrom, J.-E., Lambort, B. & Ringborg, U. (1969). J. Mol. Biol. 44, 517-532. Egyhazi, E., Daneholt, B., Edstrom, J.-E., Lambert, B. & Ringborg, U. (1970). J. Cell BioZ. 47, 516520. Egyhazi, E., D’Monte, B. & Edstrom, J.-E. (1972). J. Cell BioZ. 53, 523-531. Ellem, K. A. 0. & Rhode, S. L. (1970). B&him. Biophys. Acta, 209, 415-425. Goldberg, I. H., Rabinowitz, M. & Reich, E. ( 1962). Proc. Nut. Acud. Sci., U.S.A. 48, 2094-2101. Harbers, E. & Miiller, W. (1962). Biochem. Biophys. Res. Commun. 7, 107-110. Hyman, R. W. & Davidson, N. ( 1970). J. Mol. BioZ. 50, 421-438. Jacob, S. T., Sajdel, E. M. & Munro, H. N. (1970). Nature (London), 225, 60-62. Kedinger, C., Gniazdowski, M., Mandel, J. L., Jr, Gissinger, F. 85 Chambon, P. (1970). Biochem. Biophys. Rec. Commun. 38, 165-171. 187, 60-61. Kersten, W., Kersten, H. & Rauen, H. M. (1960). Nature (London), Klenow, H. (1963). Biochim. Biophys. Actu, 76, 354-365. Lindell, T. J., Weinberg, E., Morris, P. W., Roeder, R. G. & Rutter, W. J. (1970). Science,
170,447-449. Maitra, U., Nakata, Y. & Hurwitz, J. (1967). J. Bill. Chem. 242, 49084918. Mosteller, R. D. & Yanofsky, C. (1970). J. Mol. BioZ. 48, 525531. Pato, M. L. & von Meyenburg, K. (1970). Cold S+ng Harbor Symp. Qua&. BioZ. 35, 497-
604. Ringborg, U. & Rydlander, L. (1971). J. Cell BioZ. 51, 355-368. Roeder, R. G. & Rutter, W. J. (1970). Proc. Nat. Acad. Sci., U.S.A. 65, 675-682. Seifart, K. H. k Sekeris, C. E. (1969). 2. Naturjwech. 24b, 1538-1544. Serlhng, E., Wobus, U. & Panitz. R ‘1972). PEBS Letters, 20, 148-153.
INITIATION
INHIBITOR
OF CHROMOSOMAL
RNA
Sippel, A. & Hartmann, G. (1968). Biochk &o&s. Acta, 157, 218-220. Stirpe, F. & Fiume, L. (1967). Biochem. J. 105, 779-782. Tamm, I., Nemes, M. M. & Osterhout, S. (1960). J. Ezp. Med. 111, 339-349. Wehrli, W., Nuesch, J., Knusel, F. & Staehelin, M. (1968). Biochim. Bhphys. 215-217. Wu, R. S. t Soeiro, R. (1971). J. Mol. Biol. 58, 481-489.
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A Tentative Initiation Inhibitor of Chromosomal Heterogeneous RNA Synthesis ENDREEQYHAZI Department of Histology Karolinska Institutet S-10401, Stockholm 60, Sweden (Received 9 July 1973, and in revised form 10 November 1973) The nucleoside analogue 5,6-dichloro-l-6-n-ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus ten&w but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRBt was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (7~5 100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When a-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulsechase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary glandcells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.
1. Introduction Several chemically distinct groups of antibiotics have been discovered which inhibit DNA-directed RNA synthesis. RNA is produced in reactions involving template DNA, polymerizing enzymes and nucleoside triphosphates, and RNA synthesis can, therefore, be interfered with at the level of each of these components. The best characterized inhibitor of RNA synthesis is actinomycin D, which binds to DNA and thereby disrupts its role as a template (Kersten et al., 1960; Goldberg et al., 1962; Harbers & Mtiller, 1962). Bacterial DNA-dependent RNA polymerases are effectively blocked by the antibiotic rifampicin (Wehrli et al., 1968; Jacob et al., 1970; Chambon et al., 1970). The best established inhibitor of a eukaryotic polymerase is the mushroom poison u-amanitin, which selectively inhibits the nucleoplasmic polymerase II, while the nucleoplasmic polymerase III and the nucleolar polymerase I are resistant towards inhibition by u-amanitin (Stirpe & Fiume, 1967; Lindell et al., 1970; Roeder & Rutter, 1970). Both u-amanitin and rifampicin exert their inhibitory effects by binding to the RNA polymerases. Furthermore, RNA synthesis can be t Abbreviations used: DRB, geneous nuclear RNA.
6,0-diohloro-l-B-n-ribof~anosylbenzimidazole;
173
HnRNA,
hetero-
174
E. EGYHAZI
suppressed by nucleoside analogues, which usually exert their inhibitory action by interfering with the intracellular nucleoside triphosphate pools. Both the template inhibitor, actinomycin D (Maitra et al., 1967; Hyman 8z Davidson, 1970), and the polymerase II inhibitor, a-amanitin (Seifart & Sekeris, 1969; Chambon et al., 1970; Jacob et al., 1970; Kedinger et al., 1970), interfere with the elongation of the growing RNA chain, although by different mechanisms. By contrast, rifampicin, the inhibitor of prokaryotic polymerases, acts on the initiation of RNA transcription but has no effect on the propagation of nascent RNA chains (Sippel &, Hartmann, 1968; Mosteller & Yanofsky, 1970). No inhibitor of any nuclear RNA synthesis has been described that acts at the level of initiation. The effect of the purine nucleoside analogue, 5,6-dichloro-I-/3-n-ribofuranosylbenzimidazole, on RNA synthesis in explanted salivary glands of Chironomus tentans has been tested earlier (Egyhazi et al., 1970; Edstrom et al., 1971). These analyses showed that DRBt selectively inhibits synthesis of chromosomal heterogeneous high molecular weight RNA. In similar experiments the effect of a-amanitin was investigated (Egyh4zi et a+?.,1972). Gel electrophoretic data revealed that the polymerase inhibitor a-amanitin, like the nucleoside analogue DRB, selectively abolishes the labelling of chromosomal HiiRNA. While a-amanitin inhibits the elongation of growing RNA chains the mechanism of inhibition by DRB is unknown. It was therefore decided to study further the inhibitory effect of DRB on synthesis of HnRNA. a-Amanitin and actinomycin D were used for comparison due to their well established effects on RNA chain propagation. The data reported here show that DRB suppresses labelling of chromosomal HnRNA by a mechanism different from that of a-amanitin and actinomycin D. This finding, in conjunction with the analogy between DRB and rifampicin as expressed in the sequence of inhibition, suggests that the nucleoside analogue inhibits HnRNA synthesis at the level of initiation.
2. Materials and Methods (a) Biological material and labelling conditions Late-fourth larval instar animals of the species Chironcrmus tentans were used, cultured as described by Beermann (1952). In each experiment 4 animals were used. Four salivary glands were explanted into 50 ~1 of modifled Cannon’s medium (Cannon, 1964; Ringborg & Rydlander, 1971) supplied with 100 &?i of [3H]cytidine and 100 PCi of [3H]uridine (28 Ci/mmol and 25 Ci/mmol, respectively) and with actinomycin D (10 pg/ml), a-amanitin (40 pg/ml), or DRB (20 pg/ml). The sister glands were incubated in a similar way but without the drugs (control glan&). As a rule, incubations were carried out for 45 and 90 mm at 18°C. Actinomycin D and DRB were obtained from Merck, Sharp and Dohme while a-amanitin wig a gift from Dr T. Wieland. (b) Isolation of chromoeomea After incubation, the glands were fixed in ethanol/acetic acid (3 : 1) at 4°C for 30 mm and rinsed 3 times for 10 min each in 70% ethanol at 4’C, and finally transferred to a mixture of ethanol/glycerol (1: 1) for 60 min at 4°C. The glands were then dissected under liquid parafikr, according to Edsttim (1964). Chromosomes I to III from 24 cells (4 glands) were pooled. (c) Extra&ion of RNA The pooled chromosomal sample was digested for 5 to 10 min in hanging drops in the liquid paraffin environment with a preincubated solution of O-02 M-Tris*HCl buffer, pH t SW footnote
on p. 173.
INITIATION
INHIBITOR
OF CHROMOSOMAL
RNA
176
7.4, containing sodium dodecyl sulphate (5 mg/m.l) and nuclease-free pronase (1 mg/ml) (Calbiochem). The dissolved sample was then transferred by means of a small piece of filter paper to 20 (~1 of E buffer (0.02 M-Tris~HCl buffer, pH 8.0, 0.02 M-NaCl, 0.002 MEDTA and 2 mg sodium dodecyl sulphate/ml), and 30 ag of Eschetichia coli RNA was added as marker (Egyhazi et al., 1969). As a rule, electrophoresis was performed immediately after extraction. (d) Electrophoresis
and rneasuremeti
of radioactivity
The RNA digest was subjected to electrophoresis in 1 y0 agarose gels. The gel slabs were prepared in E buffer and the electrophoresis was carried out as described by Daneholt (1973). At the end of the run, the gel was sliced and the slices were then transferred to Packard scintillation vials, each containing 10 ml of toluene scintillator (which in 1 1
contains
30 ml soluene (Packard),
ethanol), incubated overnight spectrometer at an efficiency
5.5 g of Permablend
at 37°C and &rally of around 33%.
counted
(Packard) in a Packard
and 20 ml methoxyliquid-scintillation
3. Results (a) Labelling
of chromosomd
RNA
in the absence of inhibitors
The electrophoretic analyses of labelled RNA from chromosomes I to III after 45 and 90 minutes of incubation with tritiated nucleosides are presented in Figure 1.
The radioactivity profile of 45-minute labelled chromosomes shows a bimodal distribution; there is a peak in the 4-8 S range and label with a variable, heterogeneous distribution
in the E-100
S range as reported
earlier
(Daneholt
et al., 1969). When
4s
I 2c
I 30
I 40
Slice no.
FIG. 1. Electrophoretic separations of chromosomal RNA after 46 and 90 min of labelling. Four glands from 4 different animals were incubated in 60 al of incubation medium oontaining 100 8Ci each of cytidine and of uridine, and their sister glands were plaoed in another 60 81 of the same medium. The glands were incubated at 18’C for 46 min and 90 min, respectively. After incubation the glands were fixed and from each gland 6 sets of chromosomes I to III were isolated by dissection. The labelled RNA from each sample was released by pronase-sodium dodecyl snlphate treatment, electrophoresis was carried out in 1 oh agarose gel and the radioactivity wes counted in a Packard liquid-scintillation spectrometer. E. coli RNA was used as marker (23 S, 16 S and 4 S). The positions of 76 S and 30 S were determined in parallel analyses of Balbiani ring 2 RNA and nucleolar RNA, respectively. For more details see Materials and Methods. -a-e--, 46-Min labelling; -O-O-, 90-min labelling.
176
E. EGYHAZI
the time of incubation was prolonged to 90 minutes the migration pattern of chromosomal RNA showed a parallel increase of radioactivity over the entire size spectrum. The RNA of chromosome IV, dominated by Balbiani ring 2 RNA, possesses labelling kinetics different from those of chromosomes I to IH, and, therefore, the present analyses of chromosomal RNA will not include RNA from chromosome IV. The finding that HnRNA has not acquired maximum labelling after 45 minutes, and that there is a further increase in the radioactivity pattern between 45 and 90 minutes, may have different reasons : (1) a slow saturation of chromosomal nucleotide pools with labelled nucleosides; (2) accumulation of finished HnRNA on the chromosomes ; (3) the time required for the completion of larger HnRNA molecules is longer than 45 minutes. This latter alternative is unlikely because it would imply a shift in the pattern after 90 minutes labelling towards the high molecular weight range in comparison to that after 45 minutes labelling. More extensive data are required to decide this question finally. (b) Inhibition
of heterogeneous nuclear RNA
by DRB
(i) Chase type of experiments Our previous work demonstrated that DRB reduced labelling of chromosomal HnRNA, at least in the 30-100 S range, to 5 to loo/, of the normal values (Egyhazi et al., 1970). Labelling of chromosomal4-8 S RNA and nucleolar preribosomal RNA was only moderately affected. The labelling of the glands in these studies was, however, carried out after 60 minutes of preincubation in the presence of the nucleoside analogue. While this approach was useful in establishing the gross effect on synthesis of nuclear RNA of various types, more appropriate experimental conditions are achieved by the chase type of experiments, which can provide information on the sequence of inhibition of chromosomal HnRNA synthesis. The presence of DRB in the incubation medium for 45 minutes, after 45 minutes of labelling in the absence of the drug, resulted in a differential change in the radioactivity pattern of chromosomal HnRNA compared to the profile of normal RNA after 45 minutes of labelling (Fig. 2(a)). Incorporation in the 4-8 S range seems not to be affected by DRB in accordance with previous observations (Egyhazi et al., 1970). Whereas there is, during DRB treatment, a continuous RNA synthesis in the high molecular weight range (75-100 S), the label associated with the smaller size range of HnRNA (below 75 S) is reduced. The degree of decrease of radioactivity in HnRNA seems to be inversely related to the size of RNA molecules. When the chase period with DRB is prolonged to 90 minutes the residual HnRNA labelling is terminated and a considerable drop in label can be recorded also in the higher molecular weight range of the spectrum (Fig. 2(b)). By contrast, the nucleoside analogue-insensitive radioactivity in the 16-30 S region shows an increase. (ii) Residual synthesis of RNA derived from chromosomespreincubded with nudeaside analogue The data described in Figure 2 reveal that a time interval of 45 to 90 minutes is required to interrupt labelling of 75-100 S RNA. On the other hand, the uptake of label by the low molecular weight HnRNA has already decreased after 25 minutes. To illuminate further the phenomenon of differential inhibition of HnRNA labelling by DRB, another line of approach was adopted. Salivary glands were preincubated
INITIATION
(75 S)
INHIBITOR
(30s)
OF CHROMOSOMAL
16s
OO Slice (a)
RNA
I IO
I 20
I 30
40
1
no
(bl
2. Eleotrophoretic separations of chromosomal RNA after labelling in absence of DRB and after chase with DRB. In each experiment 4 glands from 4 different animals were incubated in 50 ~1 of incubation medium containing 100 &i each of cytidine and of uridine, and their sister glands were placed as controls in another 60 4 of the same medium. The glands were incubated at 18°C for 46 min. While the incubation of control glands was interrupted after this time, the labelling of the other glands proceeded for an additional 46 min (a) and 90 min (b) after addition of DRB (20 pg/ml). Six sets of chromosomes I to III were dissected from each gland. For other data see the legend to Fig. 1. -@-a--, Labelling in the absence of DRB ; -O-O-, labelling in the presence of DRB. FIG.
for a brief period in the presence of DRB before the labelled nucleosides were introduced into the incubation medium. In Figure 3 are shown the results of a Xi-minute incubation with the drug preceding a labelling period of 30 minutes in the continued presence of the drug. The control glands were subjected to the same type of treatment, but in the absence of inhibitor. A comparison between the electrophoretic profiles of drug-treated and normal chromosomes confirms the chase data, in that a residual labelling of HnRNA is still taking place, though at a decreasing rate. Furthermore, DRB affects labelling to a greater extent in the low molecular weight range (in the vicinity of 30 S) than in the range around 75 S. The 1630 S range constitutes an exception for three different reasons. (1) A relatively large peak, resistant to DRB but also to actinomycin D and RNAase, in the 1630 S range superimposes the label of HnRNA; (2) some radioactivity remains, even after prolonged pretreatment with DRB evenly distributed over the whole HnRNA spectrum; (3) accumulation of preribosomal RNA of nucleolar origin in the 1830 S range contributes to the pattern of HnRNA (Ringborg & Rydlander, 1971). If the time of preincubation was extended 12
178
E. EGYHAZI
Slice no
Fro. 3. Electrophoretic separations of chromosomal RNA labelled for 30 min in the absence and in the presence of DRB titer 15 min of preincubation. Four glands were incubated st 18’C for 16 min in 60 ~1 of incubation medium conteining DRB (20 rg/ml). They were then transferred to another 50 ~1 of the same medium contkning DRB, 100 @i each of cytidine and of uridine and incubated for 30 min at 18’C. For labbelling with tritieted nucleosides in the absence of DRB, the sister glands were used in an otherwise parallel procedure. Six sets of chromosomes I to III were dissected from each gland. For other details see the legend to Fig. 1. --@-a-, Normal cells; -c-O-, DRB-treated cells.
to 45 minutes in combination with a 45-minute labelling period, DRB inhibited labelling also in the high molecular weight range (Fig. 4). However, some radioactivity remains evenly distributed over the whole HnRNA spectrum due either to elevated background level or incomplete inhibition. (c) Control experiments (i) a-Amanitin
The ability of a-amanitin to impair the elongation of polymerase D-promoted transcription of RNA in vitro is well established (Chambon et al., 1970; Lindell et al., 1970; Roeder & Rutter, 1970). Further, it has been demonstrated that u-amanitin selectively inhibits labelling of chromosomal HnRNA in salivary gland cells of chironomids (Beermann, 1971; Serfling et al., 1972; Egyhazi et al., 1972), and as much as 5 to 10% of the normal values in the 30-100 S range in Chironomw tentans. The synthesis of 4-8 S RNA is not affected, and there is a-am&n&in-resistant label in the 16-30 S region. Thus or-amanitin is suitable as a reference inhibitor in studies with DRB. a-Amanitin in its capacity as an inhibitor of the propagation of growing RNA chains should stop, under chase conditions, the synthesis of all sensitive HnRNA fractions irrespective of the length of nascent polynucleotides. This expectation is
INITIATION
INHIBITOR
OF CHROMOSOMAL
RNA
179
Slice no.
Pm. 4. Electrophoretic separations of chromosomal RNA labelled for 46 min in the absence and in the presence of DRB after 45 min of preincubation. Four glands were incubated at 18°C for 46 min in 60 ~1 of incubation medium containing DRB (20 pg/ml). They were then transferred to another 60 ~1 of the same medium containing DRB, 100 pCi each of cytidine and of uridine and incubated for 46 min at 18°C. For labelling with tritiated nwleosides in the absence of DRB, the sister glands were used in an otherwise parallel procedure. For other details see the legend to Fig. 1. -m-e--, Norma1 cells; --O--O-, DRB-treated cells.
borne out by the results presented in Figure 5, where the electrophoretic pattern of RNA from u-amanitin-treated chromosomes is compared to that from normal 45minute labelled chromosomes. The radioactivity pattern of RNA from treated glands exhibits a generally decreased labelling in the range above 4-8 S. Incorporatjion of label in the 4-8 S region is not affected by cc-amanitin since these RNA fractions are insensitive to it. (ii) Actinomycin D Actinomycin D, known as an inhibitor of elongation of all DNA-dependent RNA synthesis (Maitra et al., 1967; Hymen & Davidson, 1970), depressed labelling of RNA in Chironomus tentans by up to 98%, at least in the 30-100 S range. Salivary glands incubated for 45 minutes with tritiated nucleosides in the absence of actinomycin D were incubated for another 45 minutes in the presence of the drug. Control glands were labelled for 45 minutes. The electrophoretic analyses display a synthesis inhibition by actinomycin D, with a concomitant decay of radioactivity in the whole HnRNA range above 30 S (not shown) as was also demonstrated by chase with uamanitin.
E. EGYHAZI
180
3
.0_111-7 Slice no.
FIG. 6. Eleotrophoretic separations of chromosomal RNA in the absence of a-amanitin and sfter a chase with cr-amanitin. In each experiment 4 glands from 4 different animals were incubated in 60 ~1 of incubation medium oontaining 100 PCi both of cytidine and of uridine, and their sister glands were placed as controls in another 60 ,ul of the same medium. The glands were incubated at 18°C for 46 min. While the incubation of control glends was interrupted after 46 min, the lebelling of other glands proceeded for another 46 min after addition of a-amanitin (40 pg/ml). Six sets of ahromosomes I to III were disseated from each gland. For other data see the legend to Fig. 1. -@-a--, L&belling in the absence of drug; -O--O-, l&belling in the presence of drug.
4. Discussion (a) Dichlororibofurctnosylbenzimidazole
preferentially inhibits the
synthesis of small heterogemus nuclear RNA
molecules
Administration of DRB after an initial l&belling period of 46 minutes diminished the radioactivity in the electrophoretic pattern starting in the lowest molecular weight region. It did, however, permit a continuous increase of l&belling in the 75-100 S range during a chase period of 45 minutes. The increased label in the 75-100 S range could be explained by the fact that HnRNA at the onset of DRB treatment had not attained maximum l&belling, as discussed above. A prolongation of the DRB chase from 45 to 90 minutes gave a substantial drop of label also in the high molecular weight range of the spectrum. This would be the expected effect if DRB preferentially interrupts the synthesis of small HnRNA molecules, and HnRNA molecules in the 75-100 S region escape inhibition during a 45-minute chase. The differential inhibition is not due to DRB-resistant HnRNA fractions with slow electrophoretic migration, since after prolong&ion of the chase or after a long enough preincubation period with DRB, labelling of all HnRNA fractions is equally abolished.
INITIATION
INHIBITOR
OF
CHROMOSOMAL
RNA
181
It appears obvious that the mechanism of inhibition of HnRNA synthesis by DRB is different from that of u-amanitin or actinomycin D. Use of these inhibitors of RNA chain propagation to depress labelling of HnRNA results only in a quantitative alteration in the radioactivity pattern. The synthesis inhibition with actinomycin D or a-amanitin is accompanied by a parallel decay of label in the entire size-range of HnRNA without any appreciable shift in the profiles from the high molecular weight range towards the region of lower S values. (b) Is DRB an RNA polymerase inhibitor in living cells? There are a number of findings which make DRB unlikely as a template inhibitor similar to, for example, actinomycin D or nogalamycin (Ellem & Rhode, 1970). The nucleoside analogue (1) allows a transient RNA labelling for a limited period of time; (2) preferentially depresses label associated with the low molecular weight range of the HnRNA pattern, but later on abolishes l&belling of molecules with higher S values also; and (3) lacks influence on labelling of 4-8 S RNA with chromosomal location, and on ribosomal RNA synthesis. For similar reasons it is unlikely that DRB should exert its inhibitory action by interfering with the intranuclear nucleotide pools, which would require extremely compartmentalized chromosomal precursor pools. Available data support the involvement of only one ribonucleotide precursor pool in the synthesis of nucleolar and nucleoplaamic RNA (Wu & Soeiro, 1971). In view of these considerations, the remaining alternative, that DRB acts as a polymerase inhibitor, is most likely. This question should be finally decided in a reconstituted system with purified RNA polymerases. In vivo the active form of DRB is, however, likely to be the triphosphate derivative as is the case with other nucleoside analogues, like cordycepin (Klenow, 1963) and tubercidin (Acs et al., 1964). (c) The interference of DRB with the initiation
of heterogeneousnuclear RNA synthesis
While a-amanitin interacts with the elongation of polymerase II-promoted RNA transcription, it is unlikely that DRB interrupts HnRNA synthesis at the level of elongation. The present electrophoretic data indeed would be inconsistent with such an interpretation. Therefore, it appears relevant to ask whether DRB is an inhibitor of chain initiation. Although no direct proof can be given at the present time, the accumulated data speak strongly for the hypothesis that DRB interferes with the initiation of HnRNA synthesis. The effect of DRB on HiiRNA synthesis resembles that of rifampicin on bacterial RNA transcription. The latter is a well established inhibitor of RNA chain! initiation by interaction with bacterial RNA polymerases, without preventing the completion of polynucleotides that are being synthesized (Pato & von Meyenburg, 1970; Doolittle & Pace, 1970). Thus the analogy in the inhibitory action between rifampicin and DRB may be summarized in two points. First, the administration of drugs shortly before incubation with labelled nucleosides is associated with a persistence of labelling at a decreased rate, followed by inhibition of further radioactivity rise. Second, formation of small RNA molecules is blocked earlier than that of large molecules. Thus the analogy in inhibitory action between DRB and the initiation inhibitor rifampicin supports the idea that DRB, like rifampicin, is an inhibitor of initiation. The molecular mechanism of inhibition by DRB is unknown. However, the finding that adenosine, but not gnanosine, is capable of blocking the inhibitory
182
E. EGYHAZI
effects of DRB (Tamm et aZ., 1960; Egyhazi, unpublished results), might in conjuction with present data justify the following working hypothesis: RNA polymerases initiating the synthesis of HnRNA are sensitive to the action of DRB by competition between DRB or a DRB metabolite and ATP in the formation of the initiation complex. Nucleoside analogue metabolite molecules bound in lieu of ATP molecules to the initiation sites then block the addition of a following nucleotide. By contrast, elongating polymerases, i.e. polymerases that have already passed the initiation step at the time of addition of DRB, would escape inhibition. The skilful technical assistance of Miss Agneta Askendal is gratefully acknowledged. I am also obliged to Miss Hannele Jansson for typing the manuscript, Mm Chana Szpiro for cultivation of larvae and Dr J. Hyde for revising the English text. This investigation was supported by the Swedish Cancer Society and Karolinska Institutet (Reservationsanslaget) . REFERENCES Acs, G., Reich, E. t Mori, M. (1964). PTOC. Nat. Acad. Sci., U.S.A. 52, 493-501. Beermann, W. (1952). Chromosoma, 5, 139-198. Beermann, W. (1971). Chromosoma, 34, 152-168. Cannon, G. B. (1964). Science, 146, 1063. Chambon, P., Gissinger, F., Mandel, J. L., Jr, Kedinger, C., Gniazdowski, M., BEMeihlac, M. (1970). Cold Spring Harbor Symp. Quant. Bill. 35, 693-707. Daneholt, B. (1972). Nature New Biol. 240, 229-232. Daneholt, B., Edstrom, J.-E., Egyhazi, E., Lambert, B. & Ringborg, U. (1969). Chromo8oma, 28, 399-417. Doolittle, W. F. & Pace, N. R. (1970). Nature (London), 228, 125-129. Edstrom, J.-E. (1964). In Methods in Cell Physiology (Prescott, D., ed.), vol. 1, pp. 417447, Academic Press, New York. Edstrom, J.-E., Egyhazi, E., Daneholt, B., Lambert, B. & Ringborg, U. (1971). Chromosoma, 35, 431-442. Egyh&zi, E., Daneholt, B., Edstrom, J.-E., Lambort, B. & Ringborg, U. (1969). J. Mol. Biol. 44, 517-532. Egyhazi, E., Daneholt, B., Edstrom, J.-E., Lambert, B. & Ringborg, U. (1970). J. Cell BioZ. 47, 516520. Egyhazi, E., D’Monte, B. & Edstrom, J.-E. (1972). J. Cell BioZ. 53, 523-531. Ellem, K. A. 0. & Rhode, S. L. (1970). B&him. Biophys. Acta, 209, 415-425. Goldberg, I. H., Rabinowitz, M. & Reich, E. ( 1962). Proc. Nut. Acud. Sci., U.S.A. 48, 2094-2101. Harbers, E. & Miiller, W. (1962). Biochem. Biophys. Res. Commun. 7, 107-110. Hyman, R. W. & Davidson, N. ( 1970). J. Mol. BioZ. 50, 421-438. Jacob, S. T., Sajdel, E. M. & Munro, H. N. (1970). Nature (London), 225, 60-62. Kedinger, C., Gniazdowski, M., Mandel, J. L., Jr, Gissinger, F. 85 Chambon, P. (1970). Biochem. Biophys. Rec. Commun. 38, 165-171. 187, 60-61. Kersten, W., Kersten, H. & Rauen, H. M. (1960). Nature (London), Klenow, H. (1963). Biochim. Biophys. Actu, 76, 354-365. Lindell, T. J., Weinberg, E., Morris, P. W., Roeder, R. G. & Rutter, W. J. (1970). Science,
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INITIATION
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Sippel, A. & Hartmann, G. (1968). Biochk &o&s. Acta, 157, 218-220. Stirpe, F. & Fiume, L. (1967). Biochem. J. 105, 779-782. Tamm, I., Nemes, M. M. & Osterhout, S. (1960). J. Ezp. Med. 111, 339-349. Wehrli, W., Nuesch, J., Knusel, F. & Staehelin, M. (1968). Biochim. Bhphys. 215-217. Wu, R. S. t Soeiro, R. (1971). J. Mol. Biol. 58, 481-489.
183
Acta,
157,