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Low molecular RNA associated with chromatin Purification and characterization of RNA that stimulates RNA synthesis
584
BIOCHIMICAET BIOPHYSICAACTA
BBA 97330
LOW MOLECULAR RNA ASSOCIATED W I T H CHROMATIN: P U R I F I C A T I O N AND C H A R A C T E R I Z A T I O N OF RNA THAT STIMULATES RNA S Y N T H E S I S TAKEHARU KANEHISA, TAKAFIARU TANAKA AND YOSH[O KANO Department of Biology, Division of Science, Kobe University, Kobe (Japan) (Received April 5th, 1972)
SUMMARY I. Two species of RNA, 5.o S and 4.5 S, have been purified from chick liver chromatin that stimulate specifically RNA synthesis in vitro with chromatin as teniplate. 2. When DNA is employed as template, both RNAs are inhibitory for RNA synthesis. With chick liver chromatin as template, such stimulation is not observed with any other RNA or polyanion such as synthetic polyribonucleotide or polyvinyl sulfate. 3- Base analysis indicates that these species of RNA have a relatively higher content of uridylic acid, and available evidence suggests that 5.o-S and 4.5-S RNA are functionally unique in regulating genetic activity.
INTRODUCTION Although a small quantity of RNA is shown to be associated with chromatin (refs I-9), its biological function is still obscure. A preceding report from this laboratory has described that an RNA fraction obtained from chick liver chromatin stimulates RNA synthesis in vitro with chromatin as template, and that this stimulation is probably due to some modification of the chromatin structure so that it can serve as template 1°. Further analysis has revealed that the RNA is resolved by gel electrophoresis into four species of RNA; two of them stimulate specifically the RNA synthesis, whereas the other two are inactive. Purification and characterization of these RNAs are described in the present report.
MATERIALSAND METHODS Puri]ication o] chromatin R N A Male white Leghorn chickens (15-16 days old), maintained on a laboratory chow ad libitum, were employed for the present studies. The liver chromatin was prepared and chromatin RNA was extracted by phenol-sodium dodecyl sulfate followed by the isoamyl alcohol-chloroform procedure as described previously 1°. The RNA fraction was treated with pancreatic deoxyribonuclease and pronase, and was Bioehim. Biophys. Aeta, 277 (1972) 584-589
CHROMATIN R N A IN TEMPLATE STIMULATION
585
further purified by chromatography on a methylated albumin column under the conditions specified earlier 1°. The RNA fractions eluted at around 0.45 M and 0.55 M NaC1 were pooled separately, and were referred to as Fraction A and B RNA, respectivelyl°. The RNA preparations thus obtained were essentially free of DNA and protein. No ribonuclease activity contaminated these preparations. Fraction B RNA, 25 #g each, was further fractionated by 16 % polyacrylamide gel electrophoresis with a constant current of 5 mA per tube for 4 h at o °C as described by Busch et al. n. The RNA species separated on the gel was extracted by homogenization with 0.05 M Tris-HC1 buffer, pH 7.6, containing o.15 M NaC1, and then treated with phenol saturated with 0.05 % sodium dodecyl sulfate.
Base analysis o/chromatin RNA One mCi of carrier-free inorganic phosphate (Japan Atomic Energy Inst.) was injected intraperitoneally into each male chicken at 20 h prior to killing. The labeled chromatin RNA was isolated as described above and was hydrolysed in 0.3 M KOH for 18 h at 37 °C. The hydrolysate was subjected to paper electrophoresis, and the radioactive nucleotides were determined by direct paper strip counting using an Aloka chromatogram scanner, Model JTC 2o2B.
Preparation o/ribosomal RNA, ribosomal 5-S RNA and uncharged tRNA Chick liver ribosomal RNA was extracted by the phenol procedure, and was subiected to either methylated albumin column chromatography or 16 % polyacrylamide gel electrophoresis. The RNA eluted at 0.7-0.8 M NaC1 (18-o and 28-S RNA) from the chromatography and the 5-S' region separated on gel were employed as ribosomal RNA and ribosomal 5-S RNA, respectively.
DNA preparation DNA was prepared from chick liver chromatin as described previously1°. The specific gravity of the main peak of DNA was 1.7o.
Assay o~ RNA synthesis The standard assay mixture (o.25 ml) contained: IO/,moles of Tris-HC1 buffer, pH 7.9; 0.25/,mole of MnCI~; I/,mole of MgC12; 3/,moles of 2-mercaptoethanol; o.I /,mole each of ATP, GTP and CTP; o.I/,mole of [I~C]UTP (15 mCi/mmole; Radiochemical Centre, Amersham, England); either IO #g of DNA or 12/,g of chromatin as DNA (1.45 in A280nm/A240 nm ratio); and either 2 units of Escherichia coli DNAdependent RNA polymerase (3000 units per mg protein) with DNA as template or 4 units of the enzyme with chromatin as template. Various RNA preparations, 0.5 #g each, were added where indicated. The reaction was started by the addition of enzyme at 37 °C, and was terminated after 20 min incubation by adding 5 ml of cold IO % trichloroacetic acid. The acid-precipitable material was collected on a Millipore filter, and the radioactivity was determined using a windowless gas flow counter, Kobe Kogyo, Model PR 123-1. One unit of the enzyme activity was defined as the amount catalyzing the incorporation of I nmole of labeled UMP into RNA per h with DNA as template. Under these conditions practically no RNA synthesis was observed unless the enzyme was added exogenously. Biochim. Biophys. Acta, 277 (1972) 584-589
T. KANEHISA et al.
586 RESULTS
Resolution o] Fraction B R N A When Fraction B RNA was subiected to electrophoresis on a polyacrylamide gel (16 %), four species of RNA were resolved as shown in Fig. i. The sedimentation coefficients (s~0w) of these RNAs were estimated to be 4.0 S, 4.2 S, 4.5 S and 5.0 S using a Hitachi analytical ultracentrifuge with sehlieren optics; relative amounts were approx. 16 : 6 : 2 : 1-2, respectively.
E II
u,os .
o.1
5.os
~.ss
/
\
fl
Fig. i. Electrophoretic profile of F r a c t i o n B R N A from chick liver c h r o m a t i n on polyacrylamide gel. The figure is a densitometric tracing of the R N A (25 jug) b y a Fujiriken densitometer, Model F D - A IV. The e x p e r i m e n t a l conditions are described in Materials a n d !V[ethods. Peaks at far left and r i g h t indicate the origin and end of the gel, respectively. Minor peaks at regions which were not specified were n o t stained materials b u t due to mechanical damages.
Base composition o/chromatin R N A The results summarized in Table I show that 5.o-S and 4.5-S RNA had a relatively higher content of uridylic acid, whereas both 4.2-S and 4.o-S RNA were rich in guanylic and cytidylic acids similarly as Fraction A RNA. TABLE I BASE COMPOSITION OF R N A FRACTIONS OBTAINED FROM CHICK LIVER CHROMATIN The e x p e r i m e n t a l conditions are described in the text. The electrophoresis was carried o u t as described b y S m i t h is on a Toyo filter p a p e r No. 5 I A for 2 h at 20 V / c m w i t h 0.05 M a m m o n i u m f o r m a t e buffer, p H 3.5- E a c h value r e p r e s e n t s an average of t w o determinations.
Fractions
AMP
G-]I4P
U~fP
CMP
AMlz'+UMP GMP+CMP
AMP+GMP U3/IP+C3IP
F r a c t i o n A RI~A F r a c t i o n B RN'A 5.o-S species 4.5-S species 4.2-S species 4.o-S species
22.6 25.o 23-7 23.8 21.8 23.5
26.1 24.o 24.2 27.3 30.5 26.5
22. 5 26.9 28.1 29.6 20.5 23.7
28.8 24.1 24.o 19.3 27.5 26.3
0.82 1.o8 1.o8 1.15 0-73 0.89
o.95 o.96 o.92 1.o4 i.io i.oo
E]/ects o/ R N A preparations on R N A synthesis with DNA or chromatin as template Table I I shows effects of various RNA preparations on RNA synthesis in vitro with either chick liver chromatin or chick liver DNA as template. Inconsistent with the previous observations 1°'12-14, with free DNA as template the RNA synthesis was inhibited by the addition of either one of the RNAs listed. However, when chromatin Biochim. Biophys. Acta, 277 (1972) 584-589
CHROMA'IIN
RNA IN TEMPLATE STIMULATION
587
TABLE II EFFECT OF VARIOUS R~A CHROMATIN AS TEMPLATE
SPECIES
ON
RNA
SYNTHESIS
WITH
CHICK LIVER
DNA OR C H I C K
LIVER
Experimental conditions are described in Materials and Methods. Values show the incorporation of [x4C]UMP (nmole). Numbers in parentheses represent % stimulation ( + ) or % inhibition (--) of the control experiments. R N A added
Template DNA
None Fraction A RNA Fraction B RNA 5.o-S species 4.5-S species 4.2-S species 4.o-S species Ribosomal RNA Ribosomal 5-S RNA Yeast tRNA*
0.69 0.64 0.57 o.41 0.55 0.62 0.64 o.6i o.61 0.62
Chromatin
(-- 7) (--17) (--41) (--2o) (--IO) (-- 7) (--12) (--12) (--IO)
0.29 0.27 0.32 0.56 o.43 0.30 0.27 0.27 0.26 0.26
(-- 7) (+to) (+92) (+48) ( + 4) (-- 7) (-- 7) (--io) (--IO)
* Uncharged.
was employed as template, Fraction B RNA slightly stimulated the reaction. Among the four RNA species obtained from Fraction B RNA, 5.o-S and 4.5-S RNA were found to be effective in stimulating the reaction; 92 % and 48 % of the original activity were increased by the addition of the respective RNA species. Neither 4.2-S nor 4.o-S RNA stimulated or inhibited the RNA synthesis. Quantitative results given in Fig. 2 again show that with chromatin as template both 5.o-S and 4.5-S RNA species stimulated the reaction, and the maximum activity was observed at the concentration of 2 ffg/ml. Apparently no stimulation was found at higher concentrations.
150f ~IOOP <
5O
~
o
._~ ,~ -50,
OO
0.'8
116 2.4 IRNA (p9/ml)
312
410
'
Fig. 2. Effect of varying amounts of RNA on RNA synthesis with chick liver chromatin as template. The chromatin preparation employed exhibited 1.47 in A,6 o nm/Azao nm ratio. The RNA synthesis was assayed as described in Materials and Methods. O - O , 5.o-S RNA; O - - -O, 4.5-S RNA; 0 - 0 , 4 .2-S RNA; O - - - 0 , 4 .o-S RNA; x - x , Fraction 13 RNA; x - - - × , yeast uncharged tRNA. Biochim. Biophys. Acta, 277 (1972) 584-589
T. KANEHISA el al.
588
The stimulation of RNA synthesis appears to be specific for 5.o-S and 4.5-S RNA, and was not observed by the other polyanions such as ribosonlal RNA, ribosomal 5-S RNA, synthetic polyribonucleotides and polyvinyl sulfate. Preliminary experiments were performed to examine whether 5.o-S and 4.5-S RNA stimulated the polymerase activity or modified the chromatin structure to serve as template in this reaction. When the chromatin was first incubated with various RNA preparations and then the reaction was initiated by the addition of polymerase, the incorporation of labeled precursor into RNA was stimulated significantly by a prior incubation with 5.o-S or 4.5-S RNA but not with the other RNA preparations. However, if polymerase was first incubated with RNA preparations and the reaction was started by the subsequent addition of template, all RNAs were found to be inhibitory similarly as Fraction B RNA ~°. In another set of experiments, the reaction was first started by incubating the enzyme and other necessary components, and then RNA preparations were added to the reaction mixture. With chromatin as template the addition of either 5.o-S or 4.5-S RNA immediately enhanced the RNA synthesis, while the other RNAs did not affect the reaction (Fig. 3A). In contrast, with free DNA as template, none of the RNA preparations caused profound inhibition (Fig. 3B). These results
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Fig. 3. E f f e c t of v a r i o u s R N A species on R N A s y n t h e s i s w i t h c h i c k l i ve r c h r o m a t i n or c h i c k l i v e r D N A as t e m p l a t e . The R N A s y n t h e s i s was a s s a y e d u n d e r t h e s t a n d a r d c o n d i t i o n s w i t h e i t h e r c h r o m a t i n (A) or D N A (B) as t e m p l a t e . A f t e r i o - m i n i n c u b a t i o n t h e R N A i n d i c a t e d , 0.5 fig each, w a s a d d e d . × - ; < , control; Q - - - Q , 5.o-S R N A ; ! O - - - O , 4.5 S R N A i - - -, c h i c k l i ve r r i b o s o m a l 5-S R N A ; &- - -, y e a s t u n c h a r g e d t R N A .
suggest that with free DNA as template all RNA preparations m a y bind polymerase and remove the enzyme which is available for the RNA polymerization reaction, whereas with chromatin as template 5.o-S and 4.5-S RNA m a y interact primarily with chromatin resulting in some modification of its structure to serve as template for the RNA synthesis.
Biochim. Biophys. ,4eta, 277 (1972) 584-589
CHROMATIN
RNA
IN TEMPLATE STIMULATION
589
DISCUSSION
An RNA species which contains a relatively higher amount of uridylic acid has been reported by several investigators ~'5,8. Prestayko and Busch 7 have also described an uracil-rich RNA obtained from rat liver chromatin which appears in the 5-S region on a Sephadex column. They have also shown another species of 5-S RNA obtained from rat liver nucleoli which has a higher content of uridylic acid than ribosomal 5-S RNA 15. The present study has shown that 5.o-S RNA associated with chick liver chromatin has a relatively higher content of uridylic acid, and is markedly different from ribosomal 5-S RNA which contains higher amounts of guanylic and cytidylic acids le. Ribosomal 5-S RNA is inactive in stimulating the RNA synthesis as described above. Although the correlation of the chromatin 5.o-S RNA to the nuclear and nucleolar 5-S RNA preparations obtained by Busch et al. ~,15 has remained obscure, the chromatin 5.o-S RNA presented in this paper appears to be a functionally unique RNA which may regulate RNA synthesis with chromatin as template. The 4.5-S RNA has also a relatively higher content of uridylic acid, and is probably related to nuclear 4.5-S RNA isolated from rat liver and Novikoff hepatoma 9'17. Both 4.2-S and 4.o-S RNA are shown to be higher in guanylic and cytidylic acids, and do not stimulate the RNA synthesis. However, no evidence has been available indicating whether the several RNA species described above exist in vivo or are produced from preexisting larger RNA molecules during the isolation procedure. The more precise nature as well as the interaction of these RNAs with chromatin will be explored by further investigations. ACKNOWLEDGEMENT
The authors are grateful to Dr Yasutomi Nishizuka, the Medical school, Kobe University, for reading this manuscript and valuable discussions. Thanks are also due to Dr Tadashi Yamamoto, Tokyo University, for valuable help in this study. This investigation has been supported in part by the research grants from the Scientific Research Fund of Education of Japan (1971) . REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 14 15 16 17 18
R. C. C. H u a n g and J. Bonner, Proc. Natl. Acad. Sci. U.S., 54 (1965) 960. T. Y. Shih and J. Bonner, Biochim. Biophys. Acta, 182 (1969) 30. Y. ]Vf. Sivolap and J. Bonner, Proc. Natl. Acad. Sci. U.S., 68 (1971) 387. J. H. Frenster, Nature, 2o6 (1965) 680. W. Benjamin, O. A. Levander, A. Gellhorn and R. H. Debellis, Proc. Natl. Acad. Sci. U.S., 55 (1966) 858. S. T. Jacob and I-L Busch, Biochim. Biophys. Acta, 138 (1967) 249. A. W. Prestayko and I-L Busch, Biochim. Biophys. Acta, 169 (I968) 317. R. J. Britten and E. H. Davidson, Science, 165 (1969) 349. S. 1VL E1-Khatib, T. S. Ro-Choi, Y. C. Choi and H. Busch, J. Biol. Chem., 245 (197 o) 3416. T. Kanehisa, H. Fujitani, M. Sano and T. Takana, Biochim. Biophys. Acta, 24 ° (1971) 46. A. W. Prestayko, N[. Tonato, B. C. Lewis and H. Busch, J. Biol. Chem., 246 (I97 I) 182. A. Tissi~res, S. Bourgeois and F. Gros, J. Mol. Biol., 7 (1963) ioo. H. Bremer, C. Yegian and M. Konrad, J. Mol. Biol., 16 (1965) 94. U. Maitra and J. Hurwitz, J. Biol. Chem., z42 (i967) 4897 . T. N a k a m u r a , A. W. P r e s t a y k o and H. Busch, J. Biol. Chem., 243 (I968) 1368. F. Galibert, C. J. Larsen, J. C. Lelong a n d M. Boiron, Bull. Soc. Chim. Biol., 48 (1966) 21. T. S. Ro-Choi, Y. Moriyama, Y. C. Choi and H. Busch, J. Biol. Chem., 245 (I97 o) 197 o. J. D. Smith, The Nucleic Acids 1, Academic Press, New York, 1955, p. 267.
Biochim. Biophys. Acta, 277 (1972) 584-589
BIOCHIMICAET BIOPHYSICAACTA
BBA 97330
LOW MOLECULAR RNA ASSOCIATED W I T H CHROMATIN: P U R I F I C A T I O N AND C H A R A C T E R I Z A T I O N OF RNA THAT STIMULATES RNA S Y N T H E S I S TAKEHARU KANEHISA, TAKAFIARU TANAKA AND YOSH[O KANO Department of Biology, Division of Science, Kobe University, Kobe (Japan) (Received April 5th, 1972)
SUMMARY I. Two species of RNA, 5.o S and 4.5 S, have been purified from chick liver chromatin that stimulate specifically RNA synthesis in vitro with chromatin as teniplate. 2. When DNA is employed as template, both RNAs are inhibitory for RNA synthesis. With chick liver chromatin as template, such stimulation is not observed with any other RNA or polyanion such as synthetic polyribonucleotide or polyvinyl sulfate. 3- Base analysis indicates that these species of RNA have a relatively higher content of uridylic acid, and available evidence suggests that 5.o-S and 4.5-S RNA are functionally unique in regulating genetic activity.
INTRODUCTION Although a small quantity of RNA is shown to be associated with chromatin (refs I-9), its biological function is still obscure. A preceding report from this laboratory has described that an RNA fraction obtained from chick liver chromatin stimulates RNA synthesis in vitro with chromatin as template, and that this stimulation is probably due to some modification of the chromatin structure so that it can serve as template 1°. Further analysis has revealed that the RNA is resolved by gel electrophoresis into four species of RNA; two of them stimulate specifically the RNA synthesis, whereas the other two are inactive. Purification and characterization of these RNAs are described in the present report.
MATERIALSAND METHODS Puri]ication o] chromatin R N A Male white Leghorn chickens (15-16 days old), maintained on a laboratory chow ad libitum, were employed for the present studies. The liver chromatin was prepared and chromatin RNA was extracted by phenol-sodium dodecyl sulfate followed by the isoamyl alcohol-chloroform procedure as described previously 1°. The RNA fraction was treated with pancreatic deoxyribonuclease and pronase, and was Bioehim. Biophys. Aeta, 277 (1972) 584-589
CHROMATIN R N A IN TEMPLATE STIMULATION
585
further purified by chromatography on a methylated albumin column under the conditions specified earlier 1°. The RNA fractions eluted at around 0.45 M and 0.55 M NaC1 were pooled separately, and were referred to as Fraction A and B RNA, respectivelyl°. The RNA preparations thus obtained were essentially free of DNA and protein. No ribonuclease activity contaminated these preparations. Fraction B RNA, 25 #g each, was further fractionated by 16 % polyacrylamide gel electrophoresis with a constant current of 5 mA per tube for 4 h at o °C as described by Busch et al. n. The RNA species separated on the gel was extracted by homogenization with 0.05 M Tris-HC1 buffer, pH 7.6, containing o.15 M NaC1, and then treated with phenol saturated with 0.05 % sodium dodecyl sulfate.
Base analysis o/chromatin RNA One mCi of carrier-free inorganic phosphate (Japan Atomic Energy Inst.) was injected intraperitoneally into each male chicken at 20 h prior to killing. The labeled chromatin RNA was isolated as described above and was hydrolysed in 0.3 M KOH for 18 h at 37 °C. The hydrolysate was subjected to paper electrophoresis, and the radioactive nucleotides were determined by direct paper strip counting using an Aloka chromatogram scanner, Model JTC 2o2B.
Preparation o/ribosomal RNA, ribosomal 5-S RNA and uncharged tRNA Chick liver ribosomal RNA was extracted by the phenol procedure, and was subiected to either methylated albumin column chromatography or 16 % polyacrylamide gel electrophoresis. The RNA eluted at 0.7-0.8 M NaC1 (18-o and 28-S RNA) from the chromatography and the 5-S' region separated on gel were employed as ribosomal RNA and ribosomal 5-S RNA, respectively.
DNA preparation DNA was prepared from chick liver chromatin as described previously1°. The specific gravity of the main peak of DNA was 1.7o.
Assay o~ RNA synthesis The standard assay mixture (o.25 ml) contained: IO/,moles of Tris-HC1 buffer, pH 7.9; 0.25/,mole of MnCI~; I/,mole of MgC12; 3/,moles of 2-mercaptoethanol; o.I /,mole each of ATP, GTP and CTP; o.I/,mole of [I~C]UTP (15 mCi/mmole; Radiochemical Centre, Amersham, England); either IO #g of DNA or 12/,g of chromatin as DNA (1.45 in A280nm/A240 nm ratio); and either 2 units of Escherichia coli DNAdependent RNA polymerase (3000 units per mg protein) with DNA as template or 4 units of the enzyme with chromatin as template. Various RNA preparations, 0.5 #g each, were added where indicated. The reaction was started by the addition of enzyme at 37 °C, and was terminated after 20 min incubation by adding 5 ml of cold IO % trichloroacetic acid. The acid-precipitable material was collected on a Millipore filter, and the radioactivity was determined using a windowless gas flow counter, Kobe Kogyo, Model PR 123-1. One unit of the enzyme activity was defined as the amount catalyzing the incorporation of I nmole of labeled UMP into RNA per h with DNA as template. Under these conditions practically no RNA synthesis was observed unless the enzyme was added exogenously. Biochim. Biophys. Acta, 277 (1972) 584-589
T. KANEHISA et al.
586 RESULTS
Resolution o] Fraction B R N A When Fraction B RNA was subiected to electrophoresis on a polyacrylamide gel (16 %), four species of RNA were resolved as shown in Fig. i. The sedimentation coefficients (s~0w) of these RNAs were estimated to be 4.0 S, 4.2 S, 4.5 S and 5.0 S using a Hitachi analytical ultracentrifuge with sehlieren optics; relative amounts were approx. 16 : 6 : 2 : 1-2, respectively.
E II
u,os .
o.1
5.os
~.ss
/
\
fl
Fig. i. Electrophoretic profile of F r a c t i o n B R N A from chick liver c h r o m a t i n on polyacrylamide gel. The figure is a densitometric tracing of the R N A (25 jug) b y a Fujiriken densitometer, Model F D - A IV. The e x p e r i m e n t a l conditions are described in Materials a n d !V[ethods. Peaks at far left and r i g h t indicate the origin and end of the gel, respectively. Minor peaks at regions which were not specified were n o t stained materials b u t due to mechanical damages.
Base composition o/chromatin R N A The results summarized in Table I show that 5.o-S and 4.5-S RNA had a relatively higher content of uridylic acid, whereas both 4.2-S and 4.o-S RNA were rich in guanylic and cytidylic acids similarly as Fraction A RNA. TABLE I BASE COMPOSITION OF R N A FRACTIONS OBTAINED FROM CHICK LIVER CHROMATIN The e x p e r i m e n t a l conditions are described in the text. The electrophoresis was carried o u t as described b y S m i t h is on a Toyo filter p a p e r No. 5 I A for 2 h at 20 V / c m w i t h 0.05 M a m m o n i u m f o r m a t e buffer, p H 3.5- E a c h value r e p r e s e n t s an average of t w o determinations.
Fractions
AMP
G-]I4P
U~fP
CMP
AMlz'+UMP GMP+CMP
AMP+GMP U3/IP+C3IP
F r a c t i o n A RI~A F r a c t i o n B RN'A 5.o-S species 4.5-S species 4.2-S species 4.o-S species
22.6 25.o 23-7 23.8 21.8 23.5
26.1 24.o 24.2 27.3 30.5 26.5
22. 5 26.9 28.1 29.6 20.5 23.7
28.8 24.1 24.o 19.3 27.5 26.3
0.82 1.o8 1.o8 1.15 0-73 0.89
o.95 o.96 o.92 1.o4 i.io i.oo
E]/ects o/ R N A preparations on R N A synthesis with DNA or chromatin as template Table I I shows effects of various RNA preparations on RNA synthesis in vitro with either chick liver chromatin or chick liver DNA as template. Inconsistent with the previous observations 1°'12-14, with free DNA as template the RNA synthesis was inhibited by the addition of either one of the RNAs listed. However, when chromatin Biochim. Biophys. Acta, 277 (1972) 584-589
CHROMA'IIN
RNA IN TEMPLATE STIMULATION
587
TABLE II EFFECT OF VARIOUS R~A CHROMATIN AS TEMPLATE
SPECIES
ON
RNA
SYNTHESIS
WITH
CHICK LIVER
DNA OR C H I C K
LIVER
Experimental conditions are described in Materials and Methods. Values show the incorporation of [x4C]UMP (nmole). Numbers in parentheses represent % stimulation ( + ) or % inhibition (--) of the control experiments. R N A added
Template DNA
None Fraction A RNA Fraction B RNA 5.o-S species 4.5-S species 4.2-S species 4.o-S species Ribosomal RNA Ribosomal 5-S RNA Yeast tRNA*
0.69 0.64 0.57 o.41 0.55 0.62 0.64 o.6i o.61 0.62
Chromatin
(-- 7) (--17) (--41) (--2o) (--IO) (-- 7) (--12) (--12) (--IO)
0.29 0.27 0.32 0.56 o.43 0.30 0.27 0.27 0.26 0.26
(-- 7) (+to) (+92) (+48) ( + 4) (-- 7) (-- 7) (--io) (--IO)
* Uncharged.
was employed as template, Fraction B RNA slightly stimulated the reaction. Among the four RNA species obtained from Fraction B RNA, 5.o-S and 4.5-S RNA were found to be effective in stimulating the reaction; 92 % and 48 % of the original activity were increased by the addition of the respective RNA species. Neither 4.2-S nor 4.o-S RNA stimulated or inhibited the RNA synthesis. Quantitative results given in Fig. 2 again show that with chromatin as template both 5.o-S and 4.5-S RNA species stimulated the reaction, and the maximum activity was observed at the concentration of 2 ffg/ml. Apparently no stimulation was found at higher concentrations.
150f ~IOOP <
5O
~
o
._~ ,~ -50,
OO
0.'8
116 2.4 IRNA (p9/ml)
312
410
'
Fig. 2. Effect of varying amounts of RNA on RNA synthesis with chick liver chromatin as template. The chromatin preparation employed exhibited 1.47 in A,6 o nm/Azao nm ratio. The RNA synthesis was assayed as described in Materials and Methods. O - O , 5.o-S RNA; O - - -O, 4.5-S RNA; 0 - 0 , 4 .2-S RNA; O - - - 0 , 4 .o-S RNA; x - x , Fraction 13 RNA; x - - - × , yeast uncharged tRNA. Biochim. Biophys. Acta, 277 (1972) 584-589
T. KANEHISA el al.
588
The stimulation of RNA synthesis appears to be specific for 5.o-S and 4.5-S RNA, and was not observed by the other polyanions such as ribosonlal RNA, ribosomal 5-S RNA, synthetic polyribonucleotides and polyvinyl sulfate. Preliminary experiments were performed to examine whether 5.o-S and 4.5-S RNA stimulated the polymerase activity or modified the chromatin structure to serve as template in this reaction. When the chromatin was first incubated with various RNA preparations and then the reaction was initiated by the addition of polymerase, the incorporation of labeled precursor into RNA was stimulated significantly by a prior incubation with 5.o-S or 4.5-S RNA but not with the other RNA preparations. However, if polymerase was first incubated with RNA preparations and the reaction was started by the subsequent addition of template, all RNAs were found to be inhibitory similarly as Fraction B RNA ~°. In another set of experiments, the reaction was first started by incubating the enzyme and other necessary components, and then RNA preparations were added to the reaction mixture. With chromatin as template the addition of either 5.o-S or 4.5-S RNA immediately enhanced the RNA synthesis, while the other RNAs did not affect the reaction (Fig. 3A). In contrast, with free DNA as template, none of the RNA preparations caused profound inhibition (Fig. 3B). These results
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10 15 TirY'e (rain)
20
Fig. 3. E f f e c t of v a r i o u s R N A species on R N A s y n t h e s i s w i t h c h i c k l i ve r c h r o m a t i n or c h i c k l i v e r D N A as t e m p l a t e . The R N A s y n t h e s i s was a s s a y e d u n d e r t h e s t a n d a r d c o n d i t i o n s w i t h e i t h e r c h r o m a t i n (A) or D N A (B) as t e m p l a t e . A f t e r i o - m i n i n c u b a t i o n t h e R N A i n d i c a t e d , 0.5 fig each, w a s a d d e d . × - ; < , control; Q - - - Q , 5.o-S R N A ; ! O - - - O , 4.5 S R N A i - - -, c h i c k l i ve r r i b o s o m a l 5-S R N A ; &- - -, y e a s t u n c h a r g e d t R N A .
suggest that with free DNA as template all RNA preparations m a y bind polymerase and remove the enzyme which is available for the RNA polymerization reaction, whereas with chromatin as template 5.o-S and 4.5-S RNA m a y interact primarily with chromatin resulting in some modification of its structure to serve as template for the RNA synthesis.
Biochim. Biophys. ,4eta, 277 (1972) 584-589
CHROMATIN
RNA
IN TEMPLATE STIMULATION
589
DISCUSSION
An RNA species which contains a relatively higher amount of uridylic acid has been reported by several investigators ~'5,8. Prestayko and Busch 7 have also described an uracil-rich RNA obtained from rat liver chromatin which appears in the 5-S region on a Sephadex column. They have also shown another species of 5-S RNA obtained from rat liver nucleoli which has a higher content of uridylic acid than ribosomal 5-S RNA 15. The present study has shown that 5.o-S RNA associated with chick liver chromatin has a relatively higher content of uridylic acid, and is markedly different from ribosomal 5-S RNA which contains higher amounts of guanylic and cytidylic acids le. Ribosomal 5-S RNA is inactive in stimulating the RNA synthesis as described above. Although the correlation of the chromatin 5.o-S RNA to the nuclear and nucleolar 5-S RNA preparations obtained by Busch et al. ~,15 has remained obscure, the chromatin 5.o-S RNA presented in this paper appears to be a functionally unique RNA which may regulate RNA synthesis with chromatin as template. The 4.5-S RNA has also a relatively higher content of uridylic acid, and is probably related to nuclear 4.5-S RNA isolated from rat liver and Novikoff hepatoma 9'17. Both 4.2-S and 4.o-S RNA are shown to be higher in guanylic and cytidylic acids, and do not stimulate the RNA synthesis. However, no evidence has been available indicating whether the several RNA species described above exist in vivo or are produced from preexisting larger RNA molecules during the isolation procedure. The more precise nature as well as the interaction of these RNAs with chromatin will be explored by further investigations. ACKNOWLEDGEMENT
The authors are grateful to Dr Yasutomi Nishizuka, the Medical school, Kobe University, for reading this manuscript and valuable discussions. Thanks are also due to Dr Tadashi Yamamoto, Tokyo University, for valuable help in this study. This investigation has been supported in part by the research grants from the Scientific Research Fund of Education of Japan (1971) . REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 14 15 16 17 18
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Biochim. Biophys. Acta, 277 (1972) 584-589