ANNUAL REVIEWS Copyright
1975.
Further
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EUKARYOTIC NUCLEAR RNA
x896
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
POLYMERASES Pierre Chambon Institut de Chimie Biologique, Facult6 de M6decine, 67085 Strasbourg, France
CONTENTS INTRODUCTION ......................... MULTIPLICITY AND NOMENCLATURE .. PURIFICATION AND PURITy ............ MOLECULAR WEIGHT ................................. . MOLECULAR STRUCTURE .......... .
Subunit Structure of Animal RNA Polymerases . . .
.
. . .
.. . . . . .
. . . . .
Immunological Properties of Animal RNA Polymerases .......................... . Possible Relationship Among Various Animal RNA Polymerases ...
Subunit Structure oj RNA Polymerases from Lower Eukaryotes ........................... .
613 614 618 618 619 620 623 624 625
INTRANUCLEAR LOCALIZATION AND FUNCTION OF THE MULTIPLE POLYMERASES
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GENERAL PROPERTIES OF EUKARYOTIC RNA POLYMERASES .... IN VITRO SELECTIVE TRANSCRIPTION BY EUKARYOTIC RNA POLYMERASES .. The Problem of Initiation of RNA Synthesis on an Intact Double Stranded DNA ...... .
Stimulatory Proteins (Factors). .
.
. . . . . . . . . . .
..
Analysis of Selectivity of Transcription on Deproteinized DNA ..... . Analysis of
Selectivity of Transcription on Chromatin ...............
.
626 628 629 630 632 632 633
INTRODUCTION
The control of transcription in nuclei of eukaryotic cells is undoubtedly a major mechanism of cellular regul a ti on (for review, see 1). Although it was conceivable that, as in prokaryotes (see 2), a single enzyme could be responsible for the synthesis of all types of cellular RNA (3), a major advance has been the discovery of multiple forms of nuclear DNA-dependent RNA polymerase which differ in their structure, localization, and function. While our present picture of the control of transcription in eukaryotic cells is largely drawn from analyses of the RNA synthesized in vivo, our understanding of the regulation of transcription at the molecular level requires studies with cell-free systems. Since space is limited, this review will concentrate primarily on some recent advances in our knowledge of the multiple animal nuclear RNA polymerases. However, some relevant properties of RNA polymerases isolated from lower eukaryotes will also be mentioned. Reviews on mammalian (4), animal 613
614
CHAMBON
(5), and eukaryotic (6) RNA polymerases should be consulted for historical back
ground and for discussions of some important properties of the enzymes that will not be discussed here.
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
MULTIPLICITY AND NOMENCLATURE
The existence of multiple RNA polymerases is supported by several lines of evidence obtained independently in several laboratories. First, multiple peaks of RNA poly merase activity were eluted by chromatography of solubilized enzyme preparations on DEAE-Sephadex (7, 8) or DEAE-('ellulose (9) columns (for other references, see 4-6, 10). Second, several classes of enzymes were distinguished according to the inhibitory effect of amanitin (9, 11-13). Third, structural analysis and immuno chemical properties of the purified enzymes firmly established the multiplicity of R NA polymerases (see below). The nomenclature of the multiple RNA polymerases is complex and confusing since different criteria have been used to classify the enzymes. One terminology (enzymes I, II, and III) was based mainly on the order of elution from DEAE Sephadex of the three enzymic activities found in a variety of cells (7, 8, 14). Two difficultiesare encountered with this nomenclature. First, additional enzyme activities were found that elute at lower salt concentrations than enzyme II. Second, the position of the peak corresponding to a given enzymic activity couid change from one type of cell to another (15, 16). Furthermore, the elution of a specific enzyme may differ on DEAE-cellulose and on DEAE-Sephadex. The other terminology of RNA polymerases is based on an unequivocal and easily determined criterion: sensitivity to amanitin (5, 6, 9, 17, 18). Enzymes of class A are not inhibited by amanitin at concentrations up to 10- 3 M (1 mg/ml), whereas enzymes of class B are inhibited by very low concentrations of amanitin (10 - 9 to 10 - 8 M) and class C enzymes are inhibited only 'at much higher amanitin concentrations (10 -5 to 10 -4 M). Table 1 lists the various animal RNA polymerase activities that have been isolated as distinct peaks after ion-exchange chromatography. The major change from our previous terminology (5, 6, 1 7) is related to the recent finding that several enzyme activities, including enzyme III, belong to class C. Enzyme AI (or 1) is defined as the class A-type enzyme activity that was highly purified from calf thymus (19, 20), mouse myeloma (21), and rat liver (22, 23). Chromatographic heterogeneity in class A RNA polymerase has been reported in rat liver nuclei (24), ascites tumor (25), Xenopus laevis (26), Drosophila (27), and HeLa cells (28, 45) after either phosphocellulose, CM-Sephadex, or DEAE-Sephadex column chromatography. Whether these additional peaks correspond to additional forms of class A enzyme can be questioned, since none have been purified and their resistance to high concentrations of amanitin was not tested except in the case of HeLa cells (28, 45). In rat liver, both class A enzyme activities appear to be of nucleolar origin (24). The mixture of class B enzymes was extensively purified from calf thymus (17, 29-31), rat liver (30, 32, 33), KB cells (34), mouse myeloma (3-
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!Jl
616
CHAMBON
chromatography (29) or polyacrylamide gel electrophoresis (32, 35) into three forms: BO (or 110), BI (or HA), and BII (or lis) (38). The BII form was further resolved by polyacrylamide gel electrophoresis into two isoenzymes, BIIa and BIIb (38). An additional enzyme peak belonging to class B (or II) has recently been isolated from X. laevis by chromatography on DEAE-Sephadex (26, 39). This enzyme peak, which is eluted before the main peak of activity is detected in whole cell extracts but not in purified nuclei, and thus may be of cytoplasmic origin, although nuclear leakage has not been ruled out. A similar enzyme peak belonging to class B was also isolated from whole HeLa cell extracts (28, 45). The first class C enzymes to be characterized were cytoplasmic RNA polymerase a ctivities in rat liver (40) and calf thymus (41). Although the rat liver class C enzyme activit y behaves as a sing le component when subjected to DEAE-cellulose chroma tography and elutes at the same place as class A activity, two enzyme peaks were obtained by chromatography on DEAE-Sephadex (42). The minor peak was eluted just after the peak of class A activity whereas the major peak was eluted at the same place as the peak of class B activity. Furthermore, the enzyme peak eluted at the higher ionic strength was also isolated from rat liver nuclei by chroma tography on DEAE-Sephadex (42). A class C-type polymerase activity with chro matographic and amanitin sensitivity similar to those of rat liver class C enzyme was also found recently in the cytoplasm of Chinese hamster kidney (CHK) cells (43, 44). In addition, two peaks of class C-type enzyme activity (CI and CII), eluted between class A and B activity, were obtained by chromatography of whole HeLa cell extracts on DEAE-Sephadex (28, 45). In X. laevis oocytes (46) a major fraction of the nuclear RNA polymerase activity appears to belong to class C. The enzyme peak was eluted as a single peak from DEAE-cellulose column at the same place as class A activity. When chromatographed on DEAE-Sephadex two broad peaks of activity wcre obtained (46), one eluted at very low ionic strength and the other at approximately the place of enzyme CIII (see below). RNA polymerase III was defined as the nuclear enzyme activity eluted after class B (or II) activity from DEAE-Sephadex columns (7, 8). Enzyme III has been found, usually as a minor component, in sea urchin (7, 8), rat liver (7, 14), amphibian oocytes and embryos (26, 39), human KB (47), and HeLa cells (28, 45). Mouse myeloma cells have recently been found to contain high levels of RNA polymerase III (48). Two chromatographic forms (IlIA and I1IB) have been purified (48, 49). Enzyme lIlA appears to be nuclear in origin, whereas enzyme IlIB is found mainly in the cytoplasm. Enzyme III was initially characterized as an amanitin-resistant enzyme (13, 26, 39,47), since it was not inhibited by amanitin concentrations which completely block class B en zyme activity. However, recent studies have shown that mouse myeloma (48) and HeLa cell (28, 45) enzymes III are completely inhibited by high concentrations of the toxin. Furthermore, the inhibition curves of enzymes III are identical to those of the class C enzymes isolated from rat liver (40, 42), x. laevis (46), HeLa cells (28, 45), and CHK cells (43, 44) (50% inhibition at approximately 30 Itg/mI). Therefore, enzyme(s) III belong to RNA polymerase class C as previously defined (5, 6, 40) and may tentatively be termed enzyme CIII as opposed to the two peaks of class C a cti vity eluted before class B activity from a
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
,
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
EUKARYOTIC NUCLEAR RNA POLYMERASES
617
DEAE-Sephadex column (28, 45). Although there i s n o doubt that, in the case of HeLa cells, enzyme CIII is distinct from the two class C enzyme peaks eluted from DEAE-Sephadex column between classes A and B, additional studies are required to assess whether the major peak of class C activity, isolated on DEAE Sephadex column from rat liver (40, 42) and CHK cell (43, 44) cytoplasms, is in fact identical to enzymes cm (48). As first demonstrated by Sergeant & Krsmanovic (47), enzyme CIII is not detected after chromatography on DEAE-cellulose. This absence was explained when it was found that class C enzymes were eluted from DEAE-cellulose at a much lower ionic strength at the same place as enzyme AI (28, 45, 48). This coincidental elution of enzymes cm and AI on DEAE-cellulose and the much higher activity of enzyme AI certainly explain why enzyme ClII was not previously detected in many tissues. The reason for this unique difference in behavior on DEAE-cellulose and DEAE Sephadex is unknown. However, the discovery that, at least in some cells, enzymes of class C represent a significant fraction of the RNA polymerase activity and chromatography together with class A enzymes on DEAE-cellulose points to the necessity of further characterizing all enzyme peaks previously identified as class A typc enzymes. More specifically, thc sensitivity of class A enzymes to high concen trations of amanitin and their behavior on DEAE-cellulose and DEAE-Sephadex columns should be systematically investigated. Such studies will certainly explain some of the discrepancies between the results published by different groups, for instance those concerning the characterization of the various polymerase activities isolated from X. laevis ooeytes (26, 39, 43, 46, 50, 1 78). In any case, the appearance of an additional peak of activity after ion-exchange chromatography may not represent a new enzyme. The new peak could correspond to a previously known enzyme, from which a subunit or a nonspecifically bound component has been dissociated. Furthermore, two peaks could also be obtained from the same enzyme if one of the subunits is present in the cell in limiting amounts. The present nomenclatures should therefore be considered merely as working tools, and it will not be possible to propose a fully satisfactory and systematic terminology before the molecular structure and the function of all the enzyme activities corresponding to the various chromatography peaks have been elucidated. The terminology currently used for the multiple RNA polymerase activities isolated from other eukaryotes was originally based on the same criteria as those used for animal enzymes. Two types of enzymes, A (or I) and B (or II), were distinguished according to their sensitivity to amanitin or their elution pattern during ion-exchange chromatography. However, the concentration of amanitin required for inhibition of class B enzymes is usually.higher by one or two orders of magnitude than that required to inhibit the animal class B enzymes (see 6). In some instances a third enzyme activity (class C or III) was isolated from yeast and lentil roots (see 6). It is unknown whether this enzyme activity actually belongs to an enzyme class similar to class C (as defined for animal RNA polymerases), since the possible inhibitory effect of very high concentrations of amanitin (up to 10-3 M) was not tested.
618
CHAMBON
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
PURIFICATION AND PURITY
Detailed study of the various RNA polymerases requires significant quantities of highly purified enzymes. Although on a weight basis the amount of RNA poly merase activity in eukaryotic cells is much lower than in prokaryotes, and despite many difficulties related to solubilization and instability problems (for discussion, see 4-6), RNA polymerases belonging to classes A (or I) and B (or II) have been purified to a considerable extent from a variety of eukaryotic cells. However, even from calf thymus, which is a tissue rich in RNA polymerase (51), only a few milligrams of pure class A or B enzymes were obtained per kilogram of tissue ( 19, 20, 29, 3 1). This yield, which is one to two orders of magnitude lower than that of the Escherichia coli RNA polymerase, illustrates one of the difficulties encountered in studying the regulation of transcription at the molecular level in eukaryotes. The purity of the most purified RNA polymerases from animal cells ( 19-21, 29, 30, 33-36) or yeast (52-55) is above 90%, as judged by electrophoretic analysis on polyacrylamide gels under nondenaturating conditions, and their specific activity is in the order of that of the purified E. coli RNA polymerase. Like many other nucleotidyltransferases, the animal RNA polymerases are metalloproteins requiring a tightly bound metal ion, possibly zinc (56). Both pure animal ( 19, 29, 34) and yeast (53,55) enzymes lack measurable DNase, RNase, and RNase H activities. In experi ments aimed at studying specific transcription in vitro the methods used for measur ing the possible DNase contamination should be very sensitive, since initiation of RNA synthesis could readily occur nonspecifically at single strand breaks (57) (see below). MOLECULAR WEIGHT
Purified AI RNA polymerase [calf thymus (18, 38), mouse myeloma (21)J, rat liver AI and All RNA polymerases (40, 58), purified class B RNA polymerases [calf thymus (18, 30, 38), rat liver (40, 58), and KB cells (34)], and class C RNA poly merase (40) sediment through glycerol or sucrose gradients at about 14-15S, faster than the E. coli core enzyme [mol wt 380,000-400, 000 (59)} Class B enzymes sediment slightly faster than A enzymes (38, 40), whereas class B and C enzymes are not resolved by centrifugation through sucrose gradients (40). These observations suggest '1 molecular weight of about 500,000. No drastic variation of the sedimenta tion rate was observed at different ionic strengths (34, 38). Molecular weights of 550,000 ± 10%, 600, 000 ± 10%, and 570,000 ± 10% were found for calf thymus AI, BI, and BIIa or BIIb, respectively, by electrophoresis in polyacrylamide gels of increasing porosity (38). At high ionic strength, yeast RNA polymerases of classes A and B sediment faster than E. coli holoenzyme (52, 60, 6 1), suggesting molecular weights in the order of 500, 000 . However, a lower molecular weight (440, 000) was found for yeast RNA polymerase B using polyacrylamide gels of graded porosity (60). At low salt con-
EUKARYOTIC NUCLEAR RNA POLYMERASES
619
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
centrations, the sedimentation constant of yeast enzymes A and B increased to 24 and 21 S, respectively (52, 60), suggesting that at low ionic strength these enzymes may form dimers like the bacterial enzyme but unlike the animal enzymes. Sedimentation studies in sucrose or glycerol gradients also indicate that the molecular weights of RNA polymerase B (or II) of Dictyostelium discoideum (62) and RNA polymerase A (or I) of Physarum polycephalum (16) are in the order of 420,000-500,000. Maize RNA polymerase IIa (amanitin sensitive) (63) and soybean RNA polymerases I (amanitin resistant) and II (amanitin sensitive) (64) also sediment at about 16S, suggesting molecular weights around 500,000. MOLECULAR STRUCTURE
The molecular structure of purified eukaryotic nuclear RNA polymerase has been investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). In some instances (38), since possible charge differences cannot be detected by SDS polyacrylamide gel electrophoresis, the molecular structure of the various enzymes was further investigated by polyacrylamide gel electrophoresis in the presence of urea. The molecular weight of the constitutive polypeptide chains was estimated from SDS polyacrylamide gel electrophoresis by comparison with the migration of markers of known molecular weights (65, 66). Although this method is accurate, its accuracy depends greatly on both the number of markers used for the calibration curve and the accuracy of the methods used for determining their molecular weights. It follows in particular that the values obtained for the large polypeptide chains are only indicative, since there are very few polypeptide chains with a molecular weight in the range of 120,000-200,000 which can be used for constructing a calibration curve. This certainly explains most of the discrepancies observed when comparing the molecular weights obtained for the various enzyme components in different laboratories. Such discrepancies, which are only related to differences in the slopes of the calibration curves, result in a systematic increase or decrease of the molecular weight estimates for all the components and should be distinguished from true discrepancies, which are indicated by nonsystematic variations, when comparing the molecular weights of the various polypeptide chains. For convenience, the polypeptide chains isolated on polyacrylamide gels in the presence of SDS were called subunits of the various enzymes. However, some of them (particularly some of the polypeptide chains of low molecular weight) could correspond to polypeptide chains tightly bound to the enzyme rather than to true subunits. Dissociation-reassociation experiments will be necessary to assess the effective role of these subunits in RNA synthesis. The molar ratios of the polypeptide chains in the various enzymes were estimated from the amount of dye adsorbed on each of thcm. For reasons discussed elsewhere (38) this method is not accurate, which certainly accounts for the differences observed when the molar ratios of the various components of the same enzyme were deter mined in different laboratories.
620
CHAMBON
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
Subunit Structure of Animal RNA Polymerases The molecular structure of calf thymus RNA polymerases AI, BI, BlIa, and BIIb has been studied in great detail (19, 29, 38, 67, 68), The overall pattern of the eUkaryotic enzymes resembles that of prokaryotic enzymes (59), since each enzyme consists of two subunits of high molecular weight and several molecular subunits of lower molecular weights (Table 2). The molar ratio of the large subunits is one, whereas it is higher than unity for some of the smaller subunits. The data (38) are consistent with subunit models of (SA1) 1 (SA2h (SA3h (SA4)1 (SA5h (SA6)2, (SB1)1 (SB3)1 (SB4)1_ 2 (SB5h (SB6h- 4, and (SB2h (SB3h (SB4)1- 2 (SB5h (SB6h -4 for calf thymus AI, BI, and BII enzymes, respectively. The molecular weights of enzymes calculated from their subunit composition are 501,000 ± 10%, 538,000 ± 10%, and 504,000 ± 10% for AI, BI, and BII enzymes, respectively. These values are in the range of the values expected from the sedimentation coefficients of the enzymes and, within the error limits, in rather good agreement with the molecular weight numbers obtained by electrophoresis in acrylamide gel of graded porosity (38). Gissinger & Chambon have recently shown (20) that it is possible to split by CM-Sephadex chromatography the purified calf thymus enzyme AI into two frac tions: a minor one, Ala, and a major one, Alb (in their order of elution). Fraction Ala, which lacks subunit SA3, is still enzymatically active on commercial calf thymus DNA, indicating that the SA3 component is not mandatory for RNA synthesis on this template. Since Ala is only a minor fraction of enzyme AI, it is presently unknown ,whether a small fradion of SA3 is actually removed from the full AI enzyme during CM-Sephadex chromatography, resulting in the appearance of Ala, or whether SA3 is in fact present in a limiting amount in vivo, resulting in two popUlations of AI molecules which would then be separated by CM-Sephadex chromatography. The calf thymus BII enzyme exists in two forms, BIIa and BIIb, which differ only in the charge of their largest subunits, SB2a and SB2b, respectively (38). The SB6 component of the class B enzymes consists in fact of two charge isomers, SB6a and SB6b (38). An additional component called SB5' was always associated with enzymes BI and BII, and polypeptide chains moving faster than subunits SA6 and SB6 were also seen on SDS gels (38). As pointed out above, reconstruction of the enzymes from their isolated polypeptides is required to ascertain whether these' additional polypeptide chains are actually part of the enzyme structure. The structure of purified calf thymus class B RNA polymerases was also analyzed to some extent by Weaver et al (30) and by Ingles (31). Although the latter author found a subunit composition suggesting the presence' of the two forms, BI and BII, the former group found only one predominant form similar to enzyme BII (for discussion, see 5 and 38). It is interesting that AI and B enzymes contain two pairs of small subunits of identical molecular weight and charges, SA5 and SB5, SA6 and SB6a, since it raises the possibility that there could be a common pool of low molecular weight subunits for AI and B enzymes. Fingerprint studies are required to ascertain that these subunits are, in fact, identical. The subunit pattern of the purified nucleolar mouse myeloma (21, 49) and rat
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
Table 2
Subunits of calf thymus RNA polymerases AI and B (19, 20, 29, 38, 67) Form Ala
Form Alb Molecular Subunit SAl
197
(l)b
SA2
126 (1)
SA3
51 (1)
SA4 SA5 SA6
Molecular
weighta
Form BIIa or BUb
Form HI
Subunit
weight a
SAl
197 (1 )
Molecular Subunit SBI
weight"
Molecular Subunit
214 (1) SB2a
}
weighta
SA2
126 (1)
44 (1)
SA4
44 (1)
SB4
34 (1-2)
SB4
34 (1-2)
25 (2)
SA5
25 (2)
SB5
25 (2)
SB5
25 (2 )
SB5'
20 (I)
SB5'
20 (1)
SB6(a+b)
16.5 (3�)
SB6(a+b)
16.5 (3�)
SB2b SB3
16.5 (2)
SA6
16.5 (2)
140 (1)
ttl
c:: � or
SB3
180 (1) 140 (2)
:.:
1975.
Further
Quick links to online content
All rights reserved
EUKARYOTIC NUCLEAR RNA
x896
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
POLYMERASES Pierre Chambon Institut de Chimie Biologique, Facult6 de M6decine, 67085 Strasbourg, France
CONTENTS INTRODUCTION ......................... MULTIPLICITY AND NOMENCLATURE .. PURIFICATION AND PURITy ............ MOLECULAR WEIGHT ................................. . MOLECULAR STRUCTURE .......... .
Subunit Structure of Animal RNA Polymerases . . .
.
. . .
.. . . . . .
. . . . .
Immunological Properties of Animal RNA Polymerases .......................... . Possible Relationship Among Various Animal RNA Polymerases ...
Subunit Structure oj RNA Polymerases from Lower Eukaryotes ........................... .
613 614 618 618 619 620 623 624 625
INTRANUCLEAR LOCALIZATION AND FUNCTION OF THE MULTIPLE POLYMERASES
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GENERAL PROPERTIES OF EUKARYOTIC RNA POLYMERASES .... IN VITRO SELECTIVE TRANSCRIPTION BY EUKARYOTIC RNA POLYMERASES .. The Problem of Initiation of RNA Synthesis on an Intact Double Stranded DNA ...... .
Stimulatory Proteins (Factors). .
.
. . . . . . . . . . .
..
Analysis of Selectivity of Transcription on Deproteinized DNA ..... . Analysis of
Selectivity of Transcription on Chromatin ...............
.
626 628 629 630 632 632 633
INTRODUCTION
The control of transcription in nuclei of eukaryotic cells is undoubtedly a major mechanism of cellular regul a ti on (for review, see 1). Although it was conceivable that, as in prokaryotes (see 2), a single enzyme could be responsible for the synthesis of all types of cellular RNA (3), a major advance has been the discovery of multiple forms of nuclear DNA-dependent RNA polymerase which differ in their structure, localization, and function. While our present picture of the control of transcription in eukaryotic cells is largely drawn from analyses of the RNA synthesized in vivo, our understanding of the regulation of transcription at the molecular level requires studies with cell-free systems. Since space is limited, this review will concentrate primarily on some recent advances in our knowledge of the multiple animal nuclear RNA polymerases. However, some relevant properties of RNA polymerases isolated from lower eukaryotes will also be mentioned. Reviews on mammalian (4), animal 613
614
CHAMBON
(5), and eukaryotic (6) RNA polymerases should be consulted for historical back
ground and for discussions of some important properties of the enzymes that will not be discussed here.
Annu. Rev. Biochem. 1975.44:613-638. Downloaded from www.annualreviews.org by University of Wisconsin - Madison on 08/01/12. For personal use only.
MULTIPLICITY AND NOMENCLATURE
The existence of multiple RNA polymerases is supported by several lines of evidence obtained independently in several laboratories. First, multiple peaks of RNA poly merase activity were eluted by chromatography of solubilized enzyme preparations on DEAE-Sephadex (7, 8) or DEAE-('ellulose (9) columns (for other references, see 4-6, 10). Second, several classes of enzymes were distinguished according to the inhibitory effect of amanitin (9, 11-13). Third, structural analysis and immuno chemical properties of the purified enzymes firmly established the multiplicity of R NA polymerases (see below). The nomenclature of the multiple RNA polymerases is complex and confusing since different criteria have been used to classify the enzymes. One terminology (enzymes I, II, and III) was based mainly on the order of elution from DEAE Sephadex of the three enzymic activities found in a variety of cells (7, 8, 14). Two difficultiesare encountered with this nomenclature. First, additional enzyme activities were found that elute at lower salt concentrations than enzyme II. Second, the position of the peak corresponding to a given enzymic activity couid change from one type of cell to another (15, 16). Furthermore, the elution of a specific enzyme may differ on DEAE-cellulose and on DEAE-Sephadex. The other terminology of RNA polymerases is based on an unequivocal and easily determined criterion: sensitivity to amanitin (5, 6, 9, 17, 18). Enzymes of class A are not inhibited by amanitin at concentrations up to 10- 3 M (1 mg/ml), whereas enzymes of class B are inhibited by very low concentrations of amanitin (10 - 9 to 10 - 8 M) and class C enzymes are inhibited only 'at much higher amanitin concentrations (10 -5 to 10 -4 M). Table 1 lists the various animal RNA polymerase activities that have been isolated as distinct peaks after ion-exchange chromatography. The major change from our previous terminology (5, 6, 1 7) is related to the recent finding that several enzyme activities, including enzyme III, belong to class C. Enzyme AI (or 1) is defined as the class A-type enzyme activity that was highly purified from calf thymus (19, 20), mouse myeloma (21), and rat liver (22, 23). Chromatographic heterogeneity in class A RNA polymerase has been reported in rat liver nuclei (24), ascites tumor (25), Xenopus laevis (26), Drosophila (27), and HeLa cells (28, 45) after either phosphocellulose, CM-Sephadex, or DEAE-Sephadex column chromatography. Whether these additional peaks correspond to additional forms of class A enzyme can be questioned, since none have been purified and their resistance to high concentrations of amanitin was not tested except in the case of HeLa cells (28, 45). In rat liver, both class A enzyme activities appear to be of nucleolar origin (24). The mixture of class B enzymes was extensively purified from calf thymus (17, 29-31), rat liver (30, 32, 33), KB cells (34), mouse myeloma (3-
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til :>-
�
�
!Jl
616
CHAMBON
chromatography (29) or polyacrylamide gel electrophoresis (32, 35) into three forms: BO (or 110), BI (or HA), and BII (or lis) (38). The BII form was further resolved by polyacrylamide gel electrophoresis into two isoenzymes, BIIa and BIIb (38). An additional enzyme peak belonging to class B (or II) has recently been isolated from X. laevis by chromatography on DEAE-Sephadex (26, 39). This enzyme peak, which is eluted before the main peak of activity is detected in whole cell extracts but not in purified nuclei, and thus may be of cytoplasmic origin, although nuclear leakage has not been ruled out. A similar enzyme peak belonging to class B was also isolated from whole HeLa cell extracts (28, 45). The first class C enzymes to be characterized were cytoplasmic RNA polymerase a ctivities in rat liver (40) and calf thymus (41). Although the rat liver class C enzyme activit y behaves as a sing le component when subjected to DEAE-cellulose chroma tography and elutes at the same place as class A activity, two enzyme peaks were obtained by chromatography on DEAE-Sephadex (42). The minor peak was eluted just after the peak of class A activity whereas the major peak was eluted at the same place as the peak of class B activity. Furthermore, the enzyme peak eluted at the higher ionic strength was also isolated from rat liver nuclei by chroma tography on DEAE-Sephadex (42). A class C-type polymerase activity with chro matographic and amanitin sensitivity similar to those of rat liver class C enzyme was also found recently in the cytoplasm of Chinese hamster kidney (CHK) cells (43, 44). In addition, two peaks of class C-type enzyme activity (CI and CII), eluted between class A and B activity, were obtained by chromatography of whole HeLa cell extracts on DEAE-Sephadex (28, 45). In X. laevis oocytes (46) a major fraction of the nuclear RNA polymerase activity appears to belong to class C. The enzyme peak was eluted as a single peak from DEAE-cellulose column at the same place as class A activity. When chromatographed on DEAE-Sephadex two broad peaks of activity wcre obtained (46), one eluted at very low ionic strength and the other at approximately the place of enzyme CIII (see below). RNA polymerase III was defined as the nuclear enzyme activity eluted after class B (or II) activity from DEAE-Sephadex columns (7, 8). Enzyme III has been found, usually as a minor component, in sea urchin (7, 8), rat liver (7, 14), amphibian oocytes and embryos (26, 39), human KB (47), and HeLa cells (28, 45). Mouse myeloma cells have recently been found to contain high levels of RNA polymerase III (48). Two chromatographic forms (IlIA and I1IB) have been purified (48, 49). Enzyme lIlA appears to be nuclear in origin, whereas enzyme IlIB is found mainly in the cytoplasm. Enzyme III was initially characterized as an amanitin-resistant enzyme (13, 26, 39,47), since it was not inhibited by amanitin concentrations which completely block class B en zyme activity. However, recent studies have shown that mouse myeloma (48) and HeLa cell (28, 45) enzymes III are completely inhibited by high concentrations of the toxin. Furthermore, the inhibition curves of enzymes III are identical to those of the class C enzymes isolated from rat liver (40, 42), x. laevis (46), HeLa cells (28, 45), and CHK cells (43, 44) (50% inhibition at approximately 30 Itg/mI). Therefore, enzyme(s) III belong to RNA polymerase class C as previously defined (5, 6, 40) and may tentatively be termed enzyme CIII as opposed to the two peaks of class C a cti vity eluted before class B activity from a
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,
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EUKARYOTIC NUCLEAR RNA POLYMERASES
617
DEAE-Sephadex column (28, 45). Although there i s n o doubt that, in the case of HeLa cells, enzyme CIII is distinct from the two class C enzyme peaks eluted from DEAE-Sephadex column between classes A and B, additional studies are required to assess whether the major peak of class C activity, isolated on DEAE Sephadex column from rat liver (40, 42) and CHK cell (43, 44) cytoplasms, is in fact identical to enzymes cm (48). As first demonstrated by Sergeant & Krsmanovic (47), enzyme CIII is not detected after chromatography on DEAE-cellulose. This absence was explained when it was found that class C enzymes were eluted from DEAE-cellulose at a much lower ionic strength at the same place as enzyme AI (28, 45, 48). This coincidental elution of enzymes cm and AI on DEAE-cellulose and the much higher activity of enzyme AI certainly explain why enzyme ClII was not previously detected in many tissues. The reason for this unique difference in behavior on DEAE-cellulose and DEAE Sephadex is unknown. However, the discovery that, at least in some cells, enzymes of class C represent a significant fraction of the RNA polymerase activity and chromatography together with class A enzymes on DEAE-cellulose points to the necessity of further characterizing all enzyme peaks previously identified as class A typc enzymes. More specifically, thc sensitivity of class A enzymes to high concen trations of amanitin and their behavior on DEAE-cellulose and DEAE-Sephadex columns should be systematically investigated. Such studies will certainly explain some of the discrepancies between the results published by different groups, for instance those concerning the characterization of the various polymerase activities isolated from X. laevis ooeytes (26, 39, 43, 46, 50, 1 78). In any case, the appearance of an additional peak of activity after ion-exchange chromatography may not represent a new enzyme. The new peak could correspond to a previously known enzyme, from which a subunit or a nonspecifically bound component has been dissociated. Furthermore, two peaks could also be obtained from the same enzyme if one of the subunits is present in the cell in limiting amounts. The present nomenclatures should therefore be considered merely as working tools, and it will not be possible to propose a fully satisfactory and systematic terminology before the molecular structure and the function of all the enzyme activities corresponding to the various chromatography peaks have been elucidated. The terminology currently used for the multiple RNA polymerase activities isolated from other eukaryotes was originally based on the same criteria as those used for animal enzymes. Two types of enzymes, A (or I) and B (or II), were distinguished according to their sensitivity to amanitin or their elution pattern during ion-exchange chromatography. However, the concentration of amanitin required for inhibition of class B enzymes is usually.higher by one or two orders of magnitude than that required to inhibit the animal class B enzymes (see 6). In some instances a third enzyme activity (class C or III) was isolated from yeast and lentil roots (see 6). It is unknown whether this enzyme activity actually belongs to an enzyme class similar to class C (as defined for animal RNA polymerases), since the possible inhibitory effect of very high concentrations of amanitin (up to 10-3 M) was not tested.
618
CHAMBON
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PURIFICATION AND PURITY
Detailed study of the various RNA polymerases requires significant quantities of highly purified enzymes. Although on a weight basis the amount of RNA poly merase activity in eukaryotic cells is much lower than in prokaryotes, and despite many difficulties related to solubilization and instability problems (for discussion, see 4-6), RNA polymerases belonging to classes A (or I) and B (or II) have been purified to a considerable extent from a variety of eukaryotic cells. However, even from calf thymus, which is a tissue rich in RNA polymerase (51), only a few milligrams of pure class A or B enzymes were obtained per kilogram of tissue ( 19, 20, 29, 3 1). This yield, which is one to two orders of magnitude lower than that of the Escherichia coli RNA polymerase, illustrates one of the difficulties encountered in studying the regulation of transcription at the molecular level in eukaryotes. The purity of the most purified RNA polymerases from animal cells ( 19-21, 29, 30, 33-36) or yeast (52-55) is above 90%, as judged by electrophoretic analysis on polyacrylamide gels under nondenaturating conditions, and their specific activity is in the order of that of the purified E. coli RNA polymerase. Like many other nucleotidyltransferases, the animal RNA polymerases are metalloproteins requiring a tightly bound metal ion, possibly zinc (56). Both pure animal ( 19, 29, 34) and yeast (53,55) enzymes lack measurable DNase, RNase, and RNase H activities. In experi ments aimed at studying specific transcription in vitro the methods used for measur ing the possible DNase contamination should be very sensitive, since initiation of RNA synthesis could readily occur nonspecifically at single strand breaks (57) (see below). MOLECULAR WEIGHT
Purified AI RNA polymerase [calf thymus (18, 38), mouse myeloma (21)J, rat liver AI and All RNA polymerases (40, 58), purified class B RNA polymerases [calf thymus (18, 30, 38), rat liver (40, 58), and KB cells (34)], and class C RNA poly merase (40) sediment through glycerol or sucrose gradients at about 14-15S, faster than the E. coli core enzyme [mol wt 380,000-400, 000 (59)} Class B enzymes sediment slightly faster than A enzymes (38, 40), whereas class B and C enzymes are not resolved by centrifugation through sucrose gradients (40). These observations suggest '1 molecular weight of about 500,000. No drastic variation of the sedimenta tion rate was observed at different ionic strengths (34, 38). Molecular weights of 550,000 ± 10%, 600, 000 ± 10%, and 570,000 ± 10% were found for calf thymus AI, BI, and BIIa or BIIb, respectively, by electrophoresis in polyacrylamide gels of increasing porosity (38). At high ionic strength, yeast RNA polymerases of classes A and B sediment faster than E. coli holoenzyme (52, 60, 6 1), suggesting molecular weights in the order of 500, 000 . However, a lower molecular weight (440, 000) was found for yeast RNA polymerase B using polyacrylamide gels of graded porosity (60). At low salt con-
EUKARYOTIC NUCLEAR RNA POLYMERASES
619
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centrations, the sedimentation constant of yeast enzymes A and B increased to 24 and 21 S, respectively (52, 60), suggesting that at low ionic strength these enzymes may form dimers like the bacterial enzyme but unlike the animal enzymes. Sedimentation studies in sucrose or glycerol gradients also indicate that the molecular weights of RNA polymerase B (or II) of Dictyostelium discoideum (62) and RNA polymerase A (or I) of Physarum polycephalum (16) are in the order of 420,000-500,000. Maize RNA polymerase IIa (amanitin sensitive) (63) and soybean RNA polymerases I (amanitin resistant) and II (amanitin sensitive) (64) also sediment at about 16S, suggesting molecular weights around 500,000. MOLECULAR STRUCTURE
The molecular structure of purified eukaryotic nuclear RNA polymerase has been investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). In some instances (38), since possible charge differences cannot be detected by SDS polyacrylamide gel electrophoresis, the molecular structure of the various enzymes was further investigated by polyacrylamide gel electrophoresis in the presence of urea. The molecular weight of the constitutive polypeptide chains was estimated from SDS polyacrylamide gel electrophoresis by comparison with the migration of markers of known molecular weights (65, 66). Although this method is accurate, its accuracy depends greatly on both the number of markers used for the calibration curve and the accuracy of the methods used for determining their molecular weights. It follows in particular that the values obtained for the large polypeptide chains are only indicative, since there are very few polypeptide chains with a molecular weight in the range of 120,000-200,000 which can be used for constructing a calibration curve. This certainly explains most of the discrepancies observed when comparing the molecular weights obtained for the various enzyme components in different laboratories. Such discrepancies, which are only related to differences in the slopes of the calibration curves, result in a systematic increase or decrease of the molecular weight estimates for all the components and should be distinguished from true discrepancies, which are indicated by nonsystematic variations, when comparing the molecular weights of the various polypeptide chains. For convenience, the polypeptide chains isolated on polyacrylamide gels in the presence of SDS were called subunits of the various enzymes. However, some of them (particularly some of the polypeptide chains of low molecular weight) could correspond to polypeptide chains tightly bound to the enzyme rather than to true subunits. Dissociation-reassociation experiments will be necessary to assess the effective role of these subunits in RNA synthesis. The molar ratios of the polypeptide chains in the various enzymes were estimated from the amount of dye adsorbed on each of thcm. For reasons discussed elsewhere (38) this method is not accurate, which certainly accounts for the differences observed when the molar ratios of the various components of the same enzyme were deter mined in different laboratories.
620
CHAMBON
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Subunit Structure of Animal RNA Polymerases The molecular structure of calf thymus RNA polymerases AI, BI, BlIa, and BIIb has been studied in great detail (19, 29, 38, 67, 68), The overall pattern of the eUkaryotic enzymes resembles that of prokaryotic enzymes (59), since each enzyme consists of two subunits of high molecular weight and several molecular subunits of lower molecular weights (Table 2). The molar ratio of the large subunits is one, whereas it is higher than unity for some of the smaller subunits. The data (38) are consistent with subunit models of (SA1) 1 (SA2h (SA3h (SA4)1 (SA5h (SA6)2, (SB1)1 (SB3)1 (SB4)1_ 2 (SB5h (SB6h- 4, and (SB2h (SB3h (SB4)1- 2 (SB5h (SB6h -4 for calf thymus AI, BI, and BII enzymes, respectively. The molecular weights of enzymes calculated from their subunit composition are 501,000 ± 10%, 538,000 ± 10%, and 504,000 ± 10% for AI, BI, and BII enzymes, respectively. These values are in the range of the values expected from the sedimentation coefficients of the enzymes and, within the error limits, in rather good agreement with the molecular weight numbers obtained by electrophoresis in acrylamide gel of graded porosity (38). Gissinger & Chambon have recently shown (20) that it is possible to split by CM-Sephadex chromatography the purified calf thymus enzyme AI into two frac tions: a minor one, Ala, and a major one, Alb (in their order of elution). Fraction Ala, which lacks subunit SA3, is still enzymatically active on commercial calf thymus DNA, indicating that the SA3 component is not mandatory for RNA synthesis on this template. Since Ala is only a minor fraction of enzyme AI, it is presently unknown ,whether a small fradion of SA3 is actually removed from the full AI enzyme during CM-Sephadex chromatography, resulting in the appearance of Ala, or whether SA3 is in fact present in a limiting amount in vivo, resulting in two popUlations of AI molecules which would then be separated by CM-Sephadex chromatography. The calf thymus BII enzyme exists in two forms, BIIa and BIIb, which differ only in the charge of their largest subunits, SB2a and SB2b, respectively (38). The SB6 component of the class B enzymes consists in fact of two charge isomers, SB6a and SB6b (38). An additional component called SB5' was always associated with enzymes BI and BII, and polypeptide chains moving faster than subunits SA6 and SB6 were also seen on SDS gels (38). As pointed out above, reconstruction of the enzymes from their isolated polypeptides is required to ascertain whether these' additional polypeptide chains are actually part of the enzyme structure. The structure of purified calf thymus class B RNA polymerases was also analyzed to some extent by Weaver et al (30) and by Ingles (31). Although the latter author found a subunit composition suggesting the presence' of the two forms, BI and BII, the former group found only one predominant form similar to enzyme BII (for discussion, see 5 and 38). It is interesting that AI and B enzymes contain two pairs of small subunits of identical molecular weight and charges, SA5 and SB5, SA6 and SB6a, since it raises the possibility that there could be a common pool of low molecular weight subunits for AI and B enzymes. Fingerprint studies are required to ascertain that these subunits are, in fact, identical. The subunit pattern of the purified nucleolar mouse myeloma (21, 49) and rat
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Table 2
Subunits of calf thymus RNA polymerases AI and B (19, 20, 29, 38, 67) Form Ala
Form Alb Molecular Subunit SAl
197
(l)b
SA2
126 (1)
SA3
51 (1)
SA4 SA5 SA6
Molecular
weighta
Form BIIa or BUb
Form HI
Subunit
weight a
SAl
197 (1 )
Molecular Subunit SBI
weight"
Molecular Subunit
214 (1) SB2a
}
weighta
SA2
126 (1)
44 (1)
SA4
44 (1)
SB4
34 (1-2)
SB4
34 (1-2)
25 (2)
SA5
25 (2)
SB5
25 (2)
SB5
25 (2 )
SB5'
20 (I)
SB5'
20 (1)
SB6(a+b)
16.5 (3�)
SB6(a+b)
16.5 (3�)
SB2b SB3
16.5 (2)
SA6
16.5 (2)
140 (1)
ttl
c:: � or
SB3
180 (1) 140 (2)
:.:
