RNA polymerase II.

ANNUAL REVIEWS

Further

Quick links to online content Annu. Rev Biochem. 1991. 60:689-715 Copyright © 1991 by Annual Reviews Inc. All rights reserved

Annu. Rev. Biochem. 1991.60:689-715. Downloaded from www.annualreviews.org by University of British Columbia on 04/19/13. For personal use only.

RNA POLYMERASE II Richard A. Young Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts

02 142 and

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

KEY WORDS:

transcription, mRNA synthesis.

CONTENTS PERSPECTIVES AND SUMMARY . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . .....

690

DEFINING THE ENZyME . . . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . .......... . . . . . . . . . . . . .. . . .... . . . . RNA Polymerase Purification . . . . ............ .... . ...................................... General Properties . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . .. . . . .

691 691 692 692 693

.

.

.

.

The Subunit Problem . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . Molecular Architecture of RNA Polymerase 11.............................................. YEAST RNA POLYMERASE II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. ..... . . . . . . . . . . . . . Eleven Unique Subunit Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... . .. . . . . . . .. . .. . . Subunit Features and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . Subunit Stoichiometry and Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... Subunit Interactions and RNA Polymerase Structure . . . . . . . . . . . . . . . . . .. .... ...............

693 693 695 701 702

HIGHER EliKARYOTIC RNA POLYMERASE II..... . . .. . . . . . .. . . ....... . . . . . . . . . . . . . . .. . .. Conserved Features of Subunit Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . Highly Conserved Subunit Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . ....

703 703 703

THE LARGE SUBUNIT CARBOXY TERMINAL DOMAIN . . . . . . . . . . . . . . . . . . . .. . .. . . . ...

704

Conserved Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . .. . . .... Essential Function .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... Evidence for a Role in Initiation . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... . . .. . . . ... General Models for crD Function. . . . . . . . . . . . . .......... . . . . ACTIVATION OF RNA POLYMERASE II . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

704 706 707 711

711

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . .. . . .. . . . . . . . . .

712

.

.

.

. . .

. . . . . . .

.

. . . . .

.

689

0066-4154/9 1 /070 1 -0689$02.00

690

YOUNG


PERSPECTIVES AND SUMMARY The control of transcription in eukaryotes requires a greater repertoire of proteins than it does in prokaryotes. In prokaryotes , a single core RNA polymerase enzyme composed of three subunits is responsible for all transcription . Selective promotor recognition and other regulatory controls are conferred by the association of one or more additional polypeptides with the core polymerase ( 1-8) . By contrast, eukaryotic cells contain three nuclear DNA-dependent RNA polymerases that transcribe different sets of genes. RNA polymerase I (or A) synthesizes rRNA precursors , RNA polymerase II (or B) transcribes pre-mRNA, and RNA polymerase III (or C) transcribes 5S rRNA and tRNA genes. The three eukaryotic nuclear RNA poJymerases are each composed of 8-14 polypeptides. Each of these enzymes requires the aid of a variety of additional factors for selective promoter recognition and regulated transcription initiation (9-16). The relative number and complexity of the eukaryotic RNA polymerases and their transcription factors probably reflects the need for more elaborate controls on transcription in eukaryotes. Dramatic progress has been made during the past decade in our understand ing of the components of the mRNA transcription apparatus that are important for regulated transcription initiation. The picture that has developed, in outline, is one in which RNA polymerase 11 associates with a set of TATA associated general transcription factors at the promoter; gene-specific transcription factors bind to upstream elements (enhancers and Upstream Activating Sequences) and influence the rate of transcription initiation by interacting with components of the TATA-associated transcription com plex. The TATA-associated transcription complex has at least five com ponents , TFlIA , TFIIB, TFIID, RNA polymerase II, and TFIIE ( 1 7, 1 8, reviewed in 19), and is competent to initiate transcription accurately in vitro in the absence of regulatory factors. Upstream promoter elements and the transcription factors that bind them are largely responsible for the regula tion of transcription initiation (10-15). How signals to begin RNA synthe sis are transmitted to RNA polymerase II is not yet clear, despite some recent clues (20-27). Interest in the dialogue between transciption factors and RNA polymerase II has stimulated renewed interest in the polymerase itself. Several excellent reviews address the subject of eukaryotic nuclear RNA polymerases (19, 28-32) . However, considerable new information has come from recent molecular genetic analysis of RNA polymerase II subunit genes isolated from a variety of eukaryotic organisms. This is especially true for yeast,where the isolation of genes encoding all 1 1 RNA polymerase II subunits is now complete. In addition, new information has recently emerged on the function of the unusual carboxy terminal repeat domain (CTD) of the large subunit.


RNA POLYMERASE II

691

The conservation of many features of eukaryotic RNA polymerase II subunit stlUcture and function makes the yeast enzyme a good model [or eukaryotic RNA polymerase II. The picture of RNA polymerase II that has emerged from molecular genetic analysis of the 1 1 S. cerevisiae RNA polymerase II subunits is reviewed herein . StlUctural and functional studies of yeast RNA polymerase II genes and their products have revealed that the three largest subunits are related to the prokaryotic core RNA polymerase subunits and are largely responsible for RNA catalysis. Three smaller subunits are essential components shared by all three nuclear RNA polymerases. Two of the remaining small subunits form a dissociable subcomplex that influences the efficiency of transcription initiation in vitro. The stlUcture and function of the RNA polymerase II large subunit CTD is also reviewed here. Recent evidence indicates that the CTD is involved in the regulation of transcription initiation. DEFINING THE ENZYME RNA Polymerase Purification

Three types of DNA-dependent RNA polymerases are found in the nucleus of all eukaryotic cells, each responsible for the synthesis of different classes of RNA. Roeder & Rutter (33-35) first separated the three mammalian enzymes by DEAE-Sephadex chromatography, and named them I, II , and III according to their order of elution by increasing concentrations of ammonium sulfate. RNA polymerase I is responsible for transcription of rDNA, RNA polymerase II transcribes protein-coding genes , and RNA polymerase III transcribes the genes for small stable RNAs such as tRNA and 5S rRNA. RNA polymerases I, II , and III are sometimes also called RNA polymerases A, B, and C , respectively (28, 29) . RNA polymerase II has been studied somewhat more extensively than the other two nuclear RNA polymerases . Column chromatography has been used to purify RNA polymerase IT from a large number of eukaryotes , including Saccharomyces (32), Neurospora (36), Acanthamoeba (37), Dictyostelium (38), Physarum (39) , Caenorhabditis (40) , Drosophila (4 1 ) , Xenopus (42), mouse (43), calf thymus (44), human (45) , and a variety of plants (46--50). Descriptions of the standard purification methods can be found in Sentenac (32), and modifications that effectively minimize proteolysis have been re ported (44,. 51). Immunological methods have been developed recently that permit rapid purification of enzyme that is highly active in transcription assays in vitro (52-54). Multiple chromatographic and electrophoretic forms of RNA polymerase II are detected in purified preparations of the enzyme. These various forms generally differ in the integrity of the CTD and in the extent to which the CTD

692

YOUNG

is phosphorylated (55-57). The RNA polymerase II large subunit CTD is discussed in detail in a later section.


General Properties The enzymatic properties of the RNA polymerases have been reviewed extensively (19, 29, 31, 32). Purified RNA polymerase II is capable of template-dependent synthesis of RNA in the absence of additional factors,but is not capable of precise selective initiation at promoters. Purified RNA polymerase II can be directed to "initiate" transcription at specific sites by using oligonucleotide primers,and the ability of the enzyme to extend the 3' ends of primers has been exploited to investigate its intrinsic elongation and termination properties (reviewed in 19). The degree to which RNA polymerase II itself contributes to DNA binding, selection of the precise start site, and the response to gene-specific transcrip tion factors is not yet clear. However,certain mutations in RNA polymerase II have been observed to affect transcription in a gene-specific manner (25, 58, 59), and other mutations have been found that alter the precise transcrip tion start site (D. Hekmatpanah, R. A. Young, unpublished). These results suggest that RNA polymerase II does not play an entirely passive role in selective initiation. RNA polymerase II alone can catalyze RNA chain elongation. The average elongation rate in vitro has been estimated to be between 60 and 600 nucleo tides per minute (60-62). The rate of elongation can be influenced significant ly by transcriptional pausing (61). Pausing by RNA polymerase II appears to be involved in transcription termination (63). Additional factors may have a role in regulating the rate of pausing, and thus the rate of elongation and termination. The Subunit Problem Eukaryotic RNA polymerases have traditionally been defined as the set of proteins that copurify with transcriptional activity in assays of nonspecific initiation and elongation with heterologous templates. The prokaryotic RNA polymerases,composed of three core subunits ({3' ,(3,and 0:) and a specificity factor (iT), have been defined by reconstitution from purified subunits and by genetic analysis (1-3). The eukaryotic enzymes have not yet been reconsti tuted from purified subunits, and it is not clear whether all of the polypeptides associated with the purified enzymes are genuine subunits, as opposed to adventitiously associated polypeptides. However, molecular genetic studies of Saccharomyces RNA polymerase II subunit structure and function, de scribed below, indicate that most of the polypeptides associated with the conventionally purified enzyme have a role in transcription, and are by this criterion genuine components of the transcription apparatus.

RNA

POLYMERASE II

693

Molecular Architecture of RNA Polymerase II Three features of RNA polymerase II are conserved in eukaryotes (Figure 1). First, RNA polymerase II is generally composed of 10 ± 2 subunits. Second,


the enzyme always contains two large subunits with molecular sizes of approximately 220 and 140 kDa. Finally, RNA polymerase II contains three subunits of 14-28 kDa that are also found associated with RNA polymerases I and ill in all eukaryotes in which all three nuclear enzymes have been studied. These polypeptides are called "common" or "shared" subunits. The information presented in Figure 1 may exaggerate differences in the actual number and size of the smaller RNA polymerase II subunits found in

different t:ukaryotic organisms. The subunit compositions of the RNA polymerases were ascertained in independent laboratories, using different gel electrophoresis methods and size markers. In addition, efforts to visualize the entire range of subunit sizes on SDS-PAGE sometimes resulted in poor

resolution of the smaller subunits. As is discussed in more detail below, it is likely that most of the small subunits of yeast RNA polymerase II have conserved counterparts in the other RNA polymerase II enzymes. S. cerevisiae RNA polymerases have been more intensively studied than other eukaryotic RNA polymerases, in part because of the relative ease of genetic and biochemical investigation. The conservation of many features of eukaryotic RNA polymerase II subunit structure and function indicate that the yeast enzyme is a good prototype for higher eukaryotic RNA polymerase II,

and the ability to combine powerful molecular genetic and biochemical tools with Saccharomyces greatly facilitates investigation of the molecular mech anisms involved in gene expression.

YEAST RNA POLYMERASE II Saccharomyces RNA polymerase II is composed of II polypeptides with apparent molecular weights that range from 220 to 10 kDa. RNA polymerase II purified by conventional techniques (32) or by immunoprecipitation with monoclonal antibodies directed against specific subunits (53, 54) contains 1 0 polypeptides that are readily resolved by SDS-PAGE, and an lith protein that comigrates with the 9th largest (l2-kDa) subunit. A good deal is now known about this enzyme, both from extensive biochemical characterization and from molecular genetic analysis of RNA polymerase II subunit genes.

Eleven Unique Subunit Genes The genes that encode all 1 1 yeast RNA polymerase II subunits have been isolated and sequenced (55, 64-72; N. Woychik and R. Young, unpublished). These genes, RPBI-RPBll, were isolated by screening recombinant DNA libraries of S. cerevisiae genomic DNA with a DrosophiLa large subunit

RNA POLYMERASE II

I/) Q) 0 >-

E


0 ... as J: 0 0 as en

as ... 0 Co en 0 ... :::J Q) z

200

E

:::J

G) -

en 0 >0

C

I/) :::J Co 0 c: Q) >
-


.!!! 0

..c

:s: RPB1

200

c

en

CD ... 0 0

C :::J ..c

:::J

en

c 0

E E

0 0

>< CD

a. E

0 0 ..c :::J CJ) " -�

-

�

=-200

RPB2 -

100 90

100 90

80

80

70

70

60

60

50

50

RPB3

40

40 30

20

-

-

RPB4

[[]]]]

RPB5

-

-

RPB6

-

-

nrrm

30

20

RPB7 RPB8

[[]]]] -

-

nrrm

RPB9,11 CJ 10

-

RPB10

CJ

10

Yeast RNA polymerase II subunits can be grouped into three general types. Core subunits are relatives of the E. coli RNA polymerase core subunits fJ', p, and u. Common subunits are proteins associated with all three nuclear RNA polymerases. THe RPB4/RPB7 sUbcomplex can readily dissociate from the purified enzyme, and appears to influence the efficiency of transcription initiation. RPB9 and RPBII frequently cornigrate on SOS-PAGE.

Figure 2


Table 2

Yeast RNA polymerase II subunits

Subunit

SDS-PAGE mobility 3 (Mr x 10- )

RPBI

220

Protein mass x 10 - 3)

Stoichiometry

190

1.1

(Mr

Prokaryotic Modifications phosphate

homologue {3'

Notable sequences carboxy terminal (YSPTSPSb C-Xz-C-X6-C-Xz-H-X2-H-X2 3-C-XZ-C

RPB2

ISO

140

1.0

RPB3

45

35

2.1

RPB4

32

25

0.5

RPB5

27

25

2.0

RPB6

23

IS

0.9

RPB7

17

phosphate

{3

(a

)O

C-Xz-C-XwC-XrC

phosphate

0.5

RPBS

14

17

O.S

RPB9

13

14

2.0

C-X2-C-X1S-C-XrC

RPBIO

10

SA

0.9

C-Xz-C-G

RPBll

13

14

C-X2-C-XZ4-C-XZ-C

'limited sequence similarity

;:c Z ';>

�-< � m

�m ==

§

698

YOUNG

B

A RPB1

I -

,


0

r"

F

E •

•

,

, ,

19

10

17 10

B

C

0

E F

• •

•

•

.�, , II II

5

H

G

14

IS 13

A RPB2

II

•

C

G

11 6 9

15

6103 6104 6101

H

41053 1 S 2

1---1 100 amino acids

Nucleotldo binding region

Figure 3

Segments of RPB I and RPB2 that are similar in sequence to the prokaryotic RNA

polymerase subunits {3' and {3, respectively. The eight regions of substantial homology between RPBI protein and the {3' subunit of E. coli RNA polymerase (76, 77) and the nine regions conserved between RPB2 and the {3 subunit of E. coli RNA polymerase (67, 77) are shown as black boxes . The box with diagonal lines represents the heptapeptide repeat domain. The position of RPBI and RPB2 conditional mutations that affect RNA polymerase II function or assembly are also indicated; these mutations generally affect amino acid residues that are invariant among homologous subunits from other eukaryotes (1 48). A portion of the RPB2 subunit that is involved i n nucleotide binding has been mapped to region H (98, 99).

,

subunits of eukaryotes other than yeast have been investigated, whether by immunological cross-reactivity (75 79, 80), or by gene isolation and se quence analysis (76, 8 1 -86),the two large subunits are closely related to the corresponding subunits of yeast RNA polymerase II. The two large subunits of prokaryotic and eukaryotic RNA polymerases are functional homologues. Each of the two large subunits may contain portions of the "catalytic site" for RNA synthesis. The E. coli RNA polymerase (3' subunit alone can bind to DNA, suggesting that this subunit is directly involved in the interaction of RNA polymerase with template DNA (2,3,87). The largest subunit of eukaryotic RNA polymerase II also appears to be involved in binding DNA, as suggested by blotting and UV crosslinking experiments (88-90) and the ability of monoclonal antibodies to inhibit binding (9 1 ) . Template DNA and the nascent RNA chain can be UV cross linked to both of the largest eukaryotic subunits (88, 92); the nascent RNA chain is also crosslinked to the two largest prokaryotic subunits (93). The second largest subunit of E. coli RNA polymerase ({3) and its counterpart in eukaryotic RNA polymerases can be tagged with nucleoside triphosphate substrates (94--99), and a nucleotide affinity label has been mapped to the highly conserved homology region H of yeast RPB2 (98, 99) (Figure 3) . Highly conserved lysine and histidine residues in this region may be involved


RNA POLYMERASE II

699

in nucleotilde binding,and this region thus appears to contain a portion of the catalytic site for RNA synthesis (98, 99). Several features of RPB3 (68), the third largest yeast RNA polymerase II subunit, and the E. coli RNA polymerase a subunit ( 100) suggest that the two polypeptides may be partial homologues. The two subunits are similar in size (68,1 00),in sequence (over a portion of the molecule),in stoichiometry (both polypeptides appear to exist in two copies in the RNA polymerase molecule) (2, 54), and in their apparent roles in assembly (mutations in both subunits result in the accumulation of similar subunit subcomplexes) (2, 1 0 1 ). RPB3 and a are 3 1 8 and 330 amino acids long,respectively. When the two proteins are aligned,there is a 25-amino-acid long segment [residues 29-48 in RPB3 (68)] in which 32% of the residues are identical and all but three residues are conserved; other aligned segments of the two proteins are also conserved, albeit less strikingly. SUbcomplexes of RNA polymerase II subunits that accumulate in RPB 1 ,RPB2,and RPB3 assembly mutants correspond to those found in {3' ,{3,and a assembly mutants (2,1 0 1 ). The resemblance in size and stoichiometry,the presence of limited amino acid sequence similarity,and the shared as�;embly mutant phenotypes together suggest that RPB3 and a may play similar roles in eukaryotic and prokaryotic RNA polymerases. One of these roles may be to nucleate assembly of the enzyme. The third largest subunit of human RNA polymerase II (Figure 1 ) is a homologue of the yeast RPB3 protein ( 102). Analysis of a cDNA clone encoding the human subunit (hRPB33) revealed that nearly half of the amino acid residues of human and yeast RPB3 subunits are identical . Although the three large eukaryotic RNA polymerase II subunits share many features with the three prokaryotic core subunits,the RPB 1 subunit has an unusual feature that is not shared with its prokaryotic homologue. RPB 1 contains a CTD that consists of multiple heptapeptide repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (55,56,76,8 1 ,86,reviewed in 57). The CTD is essential for cell viability ( 1 03- 106),is highly phosphorylated in a substantial portion of the RNA polymerase II molecules in the cell (5 1 ,54, 107),and appears to be involved in the response to transcription activation at some promoters (24, 25). The CTD is discussed in more detail below . RPB5, RPB6, AND RPB8 RPB5, RPB6, and RPB8 are essential components of all three nuclear RNA polymerases (70). The RPB5,RPB6,and RPB8 proteins,with apparent molecular sizes deduced from SOS-PAGE of 27, 23, and 1 4 . 5 kDa (32), have molecular sizes pre dicted from gene sequences of 25, 1 8,and 1 6 kDa,respectively (70) (Table 2). The aberrant mobility of the RPB6 protein is apparently due to the fact that it is phosphorylated (54, 1 08). Whether isolated from RNA polymerase I, II, or Ill, the subunits RPB5, RPB6, and RPB8 are indistinguishable by SDSTHE COMMON SUBUNlTS:


700

YOUNG

polyacrylamide gel mobility, fingerprint patterns (109, 110), isoelectric point (109), and antigenic recognition (74, 75, III, 112). Although their sequences have not provided clues to the functions of the common subunits, these proteins could be involved in nuclear localization, transcriptional efficiency, or the coordinate regulation of rRNA, mRNA, and tRNA synthesis. A role in DNA binding has been suggested for RPB6, but the evidence for this is indirect (74, 112). Conditional mutations already constructed in two of the common subunit genes may help elucidate the role of these subunits in RNA polymerases I, II, and III (I l 2a ; N. Woychik, S. -M. Liao, R. A. Young, unpublished) . RNA polymerase II contains three subunits of 14-28 kDa that comigrate with subunits associated with RNA polymerases I and III in all eukaryotes in which all three nuclear enzymes have been carefully studied (36-38, 42, 49, 70) (Figure 1). Antibodies have been used to confirm that, in plants, the three common subunits are in fact identical in RNA polymerases I, II, and III (49). The RPB4 and RPB7 proteins, with appar ent molecular sizes deduced from SDS-PAGE of 32 and 16 kDa (32), have molecular sizes predicted from gene sequences of 25 and 18 kDa, respectively (69; N. Woychik and R. Young, unpublished) (Table 2). These two proteins appear to form a subcomplex within RNA polymerase II that can dissociate from purified RNA polymerase II under partially denaturing conditions (113115). The RPB4 and RPB7 subunits are not essential for mRNA synthesis in vivo, as cells lacking either or both of these proteins are viable, at least at moderate temperatures (69). RPB4-deficient cells grow slowly and are tem perature sensitive, suggesting that RNA polymerase II requires the RPB4 subunit for maximal efficiency (69). Both RPB4 and RPB7 are missing from RNA polymerase II immunoprecipitated from cells lacking RPB4 (54, 115), suggesting that RPB4 provides a crucial link between the RPB4/RPB7 com plex and the rest of the polymerase subunits. Purified RNA polymerase II lacking the RPB4/RPB7 subcomplex is in distinguishable from the normal enzyme in promoter-independent initiation! chain elongation activity in vitro (115). However, enzyme lacking the RPB41 RPB7 subcomplex exhibits only limited activity in Gal4-VP16-stimulated, promoter-directed transcription initiation assays in vitro. The addition of equimolar amounts of purified RPB4/RPB7 subcomplex to enzyme lacking these subunits substantially increases the levels of selective initiation activity (115). These results suggest that the RPB4/RPB7 subcomplex influences the efficiency of selective transcription initiation. The central 102 amino acids in RPB4 are 30% identical to residues in a portion of the prokaryotic RNA polymerase (T70 subunit (69). The function of this portion of (T70 is not yet defined and so the implications of this sequence similarity are not yet clear. THE RPB4/RPB7 SUBCOMPLEX

RNA POLYMERASE

II

701

The RPB9 subunit has an apparent molecular size deduced from SDS-PAGE of 12 kDa (32) and a molecular size predicted from gene sequences of 14 kDa (72) (Table 2). RPB9 is not necessary for mRNA catalysis but is essential for cell growth at temperature extremes (72), and probably has a role in fine tuning the efficien cy of the 1ranscription apparatus. RPB9 is unusual among RNA polymerase II subunits in that it contains two cysteine repeat motifs, C-XZ-C-X\s-C-XZ-C and C-XZ-C-XZ4-C-XZ-C, which may bind some of the zinc that is associated with the enzyme (31, 116, 117). The RPB 10 subunit has an apparent molecular size deduced from SDS PAGE of 10 kDa (32) and a predicted molecular size of 5.4 kDa (71) (Table 2). RPBlO is vital for the yeast cell despite contributing only 46 amino acid residues to an enzyme that consists of a total of 4329 residues. Antigenic evidence :suggests that RPB10 may be a fourth common subunit; however, the RPB10 protein used to prepare antibodies was not characterized for its purity, and this data is difficult to interpret (112). The functional contribution of RPB10 to the transcription apparatus is not yet understood. The observation that the 12.5-kOa SDS-PAGE band can occasionally be resolved as a doublet (53) led to the discovery that RPB9 comigrates with a second polypeptide, and this second 12.5-kOa subunit was designated RPB l l (N. Woychik and R. Young, unpublished). RPB11 has a molecular size predicted from the gene sequence of 14 kDa. Unlike RPB9, RPBII is essential for yeast cell viability.


THE THREE SMALL SUBUNITS: RPB9-RPBll

Subunit Stoichiometry and Phosphorylation The isolation of the genes for yeast RNA polymerase II subunits has allowed investigators to confirm the subunit composition of the enzyme, to estimate the relative stoichiometry of each subunit, and to ascertain which subunits are phosphorylated. Ten polypeptides are immunoprecipitated from radiolabeled yeast cell extracts containing an epitope-tagged subunit (54); as mentioned previously, the 11th subunit (RPB11) frequently comigrates with RPB9. The 10 polypeptides that are isolated by this method are identical in size and number to those obtained for yeast RNA polymerase II purified by con ventional column chromatography. Similar results are obtained when the enzyme is isolated using a monoclonal antibody directed against the CTD of the largest subunit (53). The rellative stoichiometry of the yeast RNA polymerase II subunits has been estimated by combining knowledge of the methionine content of the subunits deduced from sequence analysis and the relative extents of labeling with 35S-methionine (54, 101) (Table 2). The average RNA polymerase II molecule appears to be composed of one copy each of RPB1, 2, 6, 8, 10, and 11, and two copies of RPB3, 6, and 9. The RPB4 and 7 subunits are present at less than one copy per enzyme molecule.


702

YOUNG

The in vivo phosphorylation state of RNA polymerase II subunits has also been investigated using the immunoprecipitation approach (54) . The RPB l and RPB6 subunits are clearly phosphorylated. RPB l appears to be present in both phosphorylated and unphosphorylated forms in vivo,with approximately half of the RNA polymerase II molecules containing one form, and half the other form . Most or all of the phosphate on RPB l appears to be associated with the CTD ( 10 1 ) . It is not yet clear whether the phosphorylated form of RPB 1 is a single species or whether there is some heterogeneity in the degree of phosphorylation of the RPB I CTD. Relatively low levels of phosphate can be detected on RPB2 (54); it is conceivable that this represents phosphate that is transiently bound to the subunit as an intermediate during hydrolysis of nucleoside triphosphates . Possible roles for RPB I phosphorylation are dis cussed below . Subunit Interactions and RNA Polymerase Structure

The results of genetic studies in Saccharomyces and in Drosophila indicate that RPB 1 and RPB2 interact with one another, probably at multiple sites ( 1 1 8, 1 1 9) . In yeast,suppressors of a temperature-sensitive mutation (rpbl-l) in conserved region H of RPB 1 (Figure 3) are found clustered in conserved region I of RPB2 (118). Conversely, suppressors of a temperature-sensitive mutation (rpb2-2) in region I of RPB 2 are found both in region H and in two other regions of RPB 1 ( 1 1 8) . Genetic analysis of the two large subunits of Saccharomyces RNA polymerase I has revealed that one of the interactions between the two large subunits of all three nuclear RNA polymerases may occur through two putative zinc-binding domains that are highly conserved among RNA polymerases I, II, and III ( 1 20) . One of these putative zinc binding domains is located in conserved region A of the largest subunit, and the other immediately follows conserved region I of the second largest subunit (Figure 3). While suppressor analysis should be interpreted cautiously, it is possible that both regions A and H of the largest subunit interact with region I of the second largest subunit. Although crystals of RNA polymerase II suitable for X-ray analysis have not yet been obtained, a low-resolution structure of yeast RNA polymerase II has been determined using electron diffraction of two-dimensional crystals (53, 1 21 ) . Two-dimensional crystals of single RNA polymerase TI molecules were formed on charged lipid layers; the crystals gave electron diffraction patterns extending to about 30 angstroms (53). Much greater resolution (to 1 6 angstroms) o f RNA polymerase I I i n crystals was obtained with enzyme lacking the RPB 4/RPB7 subcomplex ( 1 2 1 ), prcsumably because the RNA polymerase molecules in the original crystals were not homogeneous,as only a portion of the enzyme molecules contain the RPB4/RPB7 subcomplex. RNA polymerase II lacking the RPB 4/RPB7 subcomplex has a 25 angstrom

RNA POLYMERASE II

703

groove that bears a striking resemblance to the E. coli RNA polymerase groove that is thought to be the DNA-binding channel ( 1 22). Future analysis of two-dimensional crystals of RNA polymerase containing modified subunits may reveal the positions of individual subunits in the enzyme, and may thus provide additional clues to subunit functions. HIGHER EUKARYOTIC RNA POLYMERASE II


Conserved Features of Subunit Architecture

RNA polymerase II enzymes purified from higher eukaryotes appear to be closely related to the S. cerevisiae enzyme. Purified by conventional column chromatography, the eukaryotic enzymes are composed of 10 ± 2 subunits (Figure 1 ). The two largest subunits have molecular sizes of approximately 220 and Jl40 kDa and,where sequences are available,the two large subunits are always closely related to the yeast RPB 1 and RPB2 subunits. In addition, where the subunit composition of all three nuclear RNA polymerases has been investigated, three subunits of molecular sizes 14--28 kDa appear to be shared by all three enzymes. Highly Conserved Subunit Sequences RPBl Nearly 40% of the amino acid residues of the largest RNA polymerase II subunit (RPB 1 ) of S. cerevisiae (55) are identical in Caenorhabditis elegans (8 1 ), Drosophila melanogaster (76), and the mouse (82), and a large fraction of the remaining residues are conserved. This subunit is a homologue of the E. coli RNA polymerase {3' subunit, and the regions that are most conserved between the yeast and the E. coli large subunit are also the most conserved when any eukaryotic RPBl subunit is compared with its prokaryot ic counterpart. With one exception noted below, all of the eukaryotic RPB 1 subunits contain the unusual CTD that consists of repeats of the heptapeptide consenSU:5 sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD repeat length tends to increase with the complexity of the eukaryote; yeast has 26 or 27 repeats, depending on the strain, and man has 52.

The gcne encoding the second largest subunit of D. melanogaster RNA polymerase II has been cloned and sequenced (85, 1 23). More than a third of the amino acid residues of the S. cerevisiae and D. melanogaster RPB2 subunits are identical . RPB2

RPB3 The isolation and sequence analysis of a cDNA clone encoding the third largest human RNA polymerase II subunit (hRPB33) has revealed that 45% of the amino acid residues of human and yeast RPB3 subunits are identical ( 1 02).

704

YOUNG


COMMON SUBUNITS (RPB5, 6, AND 8) The subunit composition of all three nuclear RNA polymerases has been determined for Saccharomyces (32), Neurospora (36), Acanthamoeba (37), Dictyostelium (38), Xenopus (42), wheat, and cauliflower (49). In each case, three polypeptides of RNA polymerases I, II, and III have identical gel mobility (Figure 1 ). These apparently shared polypeptides range from 14 to 28 kDa. A eDNA clone encoding a human homologue of Saccharomyces RPB5 has been isolated and sequenced ( 1 24). This protein, called hRPB23 or HP-23, is 30% identical to yeast RPB5. RPB4/RPB7 SUBCOMPLEX The Saccharomyces RPB4 and RPB7 subunits form a subcomplex that tends to dissociate from the enzyme and appears to influence the efficiency of initiation. It is not yet clear whether higher eukaryotic �A polymerase II enzymes have components related to RPB4 and RPB7. Purified human RNA polymerase II does not contain detectable levels of an RPB4-like subunit; the fourth largest RNA polymerase II subunit of human R NA polymerase II (hRPB23) is homologous to the Saccharomyces common subunit RPB5 ( 1 24). RPB9

The yeast RPB9 subunit appears to have a homologue in D.

melanogaster, which is encoded by DNA upstream of the suppressor of Hairy

Wing gene (72, 1 25); almost 50% of the amino acid residues of the yeast and Drosophila proteins are identical. THE LARGE SUBUNIT CARBOXY TERMINAL DOMAIN This section reviews current knowledge about the structure and function of the RNA polymerase II carboxy terminal domain. The CTD is an essential component of the enzyme that appears to be necessary for normal responses to activation signals at some promoters. Conserved Structure

The RNA polymerase II large subunit CTD consists of multiple repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain does not exist in the large subunits of RNA polymerases I and III (55 , 78), nor is it found in eubacterial or archaebacterial RNA polymerases ( 1 26, 1 27) or a viral RNA polymerase ( 1 28). The CTD consensus sequence is re peated 1 7 times in Plasmodium RNA polymerase II (86), 26-27 times in Saccharomyces (55 , 1 03), 32 times in Caenorhabditis (8 1 ), 40 times in Arabadopsis ( 128a), about 45 times in Drosophila ( 1 04, 1 06), and 52 times in hamster ( 1 04) and mouse (56) (Figure 4). The CTD has an essential role in transcription; deletion mutations that remove most or all of

705

RNA POLYMERASE II Mouse

Arabadopal. Cacnorhabditls

YTPQSPS

5

4 YSpTSPS

YSPSSPN

YSPTSPG

YSPTSPS

YSPTSPL

YSPTSPT

Yeast

Plasmodium. YSPS

YSPTSPN

YEPRSPGG

YTASSPG

YSPTSPG

YGGMSPGV

FSPTSPT

YSPTSPT

GASPN

YSPTSPG

YSPSSPQ

YSPTSPA

YNANNAY

FSMTSPH

YSPTSPS

YSPTSPK

YSPSSPQ

YSPTSPS

NQNDQMNVNSQ YNVMSPV

7 YSPTSPS 8 YSPTSPS 9 YSPTSPS 10 YSPTSPS 11 YSPTSPS 12 YSPTSPS 13 YSPTSPS 14 YSPTSPS 15 YSPTSPS 16 YSPTSPS

YSPSSPA

LSPRTPS

FGVSSPG

YSPTSPA

6 YSPTSPN


Drosophila

1 2 3

YASTTPN

YSPSSPG

YSPTSPS

YSPTSPS

YSPTSPA

YSPTSPAAG

YSPTSPS

YSVTSPK

FNPNSTG

YSPTSPS

QSPVSPS

YSPTSPS

YSPTSPK

YA

SPR

YSPSSSG

YSPTSPS

YSPTSPS

YSPTSPS

YSPTSPK

YSPTSPV

YSPTSPS

YSPTSPS

YSPTSPS

YSPTSPK

YSPTVQ

YSPTSPS

YSPTSPS

YSPTSPS

YSPTSPK

FGSSPS

YSPTSPS

YSPTSPS

YSPTSPS

YSPTSPK YSPTSPK

YSPTSPS

YSPTSPS

YSPTSPS

A

YSPTSPS

YSPTSPS

YSPTSPS

YS PTSP K

YSPSSSN

YSPTSPA

YSPSSPS

YSPTSPS

YSPTSPVA QNIASPN

FAGSGSNI

YSPGN

17

YSPNSPS

YSPTSPA

YSPSSPS

YSPTSPS

YSPTSPS

18 YSPTSPS

YSPTSPS

YSPTSPA

YSPSSPR

YSPTSPS

19

YSPSSPS

YSPTSPS

YSPTSPT

YSPTSPA

YSITSPK

YSPTSPS

20 YSPTSPS

YSPTSPC

YSPTSPS

YSPTSPT

YSPTSPS

FSPTSPA

YSPTSPS

YSPTSPS

YSPTSPT

YSPTSPS

YSISSPV

YSPTSFN

YTPVTPS

YSPTSPS

YSPTSPS

YSPTSFN

YSPTSPN

YSPTSPA

YESGGG

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

YSPTSFS

YSPTSPN

YSPT SPS

YSPTSPT

YSPTSPS

YSP

YSPTSPN

YS ASPQ

YSPTSPG

YSPSSPK

Y SPT SP S

YSPTSPS

YSPASPA

YSPTSPS

YSPSSPT

YSPTSPG

YSPTSPN

YSQTGVK

YSPTSPS

YSPTSPS

YSPGSPA

YTPTSPN

YSPTSPT

YGPTSPS

YSPTSPQ

YSPKQDEQKHNENENSR

YNPQSAK

YSPTSPQ

YSPTSPS

Y S PP SP S D G

YSPGSPQ

YSP SIA

YSPSSPT

YSPTSPS

YTPGSPQ

YSPSNAR

YSPTSPT

YSPSSPR

YTPQSPT

YSPASPK YSPTSPL

LSPASP

YSPTSPS

YSPTSPN

YTPSSPQ

YTPSSPS

YSPSSPQ

YSPTSPS

YSPTSPT

YSPSSPS

HSPS

SQ

YSPTSPS

YTP SPSEQPGTSAQVDFFYSKLNF

YSPTSPK

YSPTGST

YSPSSPT

YTPTSPS

YSPTSPR

YSPSSPY

YSPSSPE

YSPNMSI

SSGASPD

YSPTSPK

YSPTSPT

YSPTLPG

YSPTSPK

YTPTARN

YTPSSTGQ

YSPTSPM

YTPHEGDKKDKTGKKDASKDDKGNP

YTPASPK

41 YSPTSPT 42 YSPTTPK

43 YSPTSPT 44 YSPTSPV 45 YTPTSPK 46 YSPTSPT 47 YSPTSPK

YSPSSTK

AA)

YSPTSPS

YTPTSFS

YSPTSPS

(+68

YSPSAG

YSPTAPSH YSPTSPA YSPSSPTFEESED

48 YSPTSPT

49 YSPTSPKGST 50 YSPTSPG 51 YSPTSPT 52 YSLTSPAISPDDSDEEN Figure 4 RNA polymerase II CTDs. CTD sequences are shown for Plasmodium (86), Saccha romyces (103), Caenorhabditis (81), Arabadopsis (l28a), Drosophila (104, 106), and mouse (56). Different yeast strains have somewhat different CTD sequences (55, 103); and that from strain S288C' (103) is shown here.


706

YOUNG

the CTD are lethal to yeast (103, 104), Drosophila (106), and murine (105) cells. The CTD is highly phosphorylated in a substantial portion of the RNA polymerase II molecules in the cell (54,107), and protein kinases have been purified that appear to be involved in CTD phosphorylation (129-131). Both the CTD repeat length and the degree of heptapeptide conservation vary from one organism to another (Figure 4). The length of the CTD generally increases with the genomic complexity of the organism. The precise heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser occurs in 3060% of the CTD repeats in Saccharomyces, Caenorhabditis, Arabadopsis, and mouse . The consensus sequence is much less well conserved in Drosophi la and in Plasmodium. Certain amino acid residues in the repeat are highly conserved in all of these organisms; in particular, the tyrosines are almost invariant . The repeat form of the CTD does not occur in all eukaryotes . The protozoan parasites Trypanosoma brucei and Crithidia Jasciculata do not contain a heptapeptide repeat structure at the carboxyl terminus of the large subunit (83,84,132),but are instead rich in the amino acid residues found in the CTDs of higher eukaryotes (serine, threonine, proline, and tyrosine). Modification and proteolysis of the CTD almost certainly accounts for most of the various forms of RNA polymerase II that have been purified from eukaryotes (51, 54-56, 107, 130, 131, 133; reviewed in 57). The largest subunit of RNA polymerase II can be resolved into three major forms in SDS-PAGE. In order of increasing mobility, these are called 110, IIa, and lIb. The 110 form of the subunit contains a highly phosphorylated CTD that can be generated in vivo or in vitro (51, 107, 108, 131, 134). The IIa form of the subunit appears to be an unmodified primary translation product (55, 56); the lIa form can be generated by phosphatase treatment of the lIo form of the subunit (107). The lIb form lacks most or all of the CTD (55,56,106),and is probably a proteolytic artifact of purification (44, 54). The CTD of the mammalian 110 form of the subunit contains phospho serine and phos phothreonine, but not detectable amounts of phosphotyrosine (107). The 110 form of the subunit is thought to predominate in HeLa cells (44), but similar amounts of the lIo form and the lIa form occur in yeast RNA polymerase II isolated with a rapid immunoprecipitation procedure (54). Essential Function

Truncation of the S. cerevisiae RNA polymerase II CTD reduces the efficien cy of a function that is essential for cell viability (103, 104, 135). RNA polymerase II large subunit genes encoding fewer than 10 complete hep tapeptide repeats are unable to sustain cell viability,those encoding 10 to 12 complete repeats produce cells that are temperature sensitive and cold sensi tive, and those encoding at least 13 complete repeats are sufficient for wild-type growth (103). Y east cells containing CTDs of 10 to 12 complete


RNA POLYMERASE II

707

repeats are also inositol auxotrophs (25, 1 35),and are unable to use pyruvate as a carbon source (D . Chao, R. A. Young, unpublished). The phenotypes associated with these CTD mutations are not a cons�quence of instability of the large subunit, as CTD truncation does not appear to alter the levels of RNA polymerase II in the cell ( 103); rather, these phenotypes appear to reflect some functional deficiency of the enzyme . The conditional phenotypes and the inositol auxotrophy suggest that the expression of some genes is more sensitive to certain CTD partial deletion mutations than are others . Truncation of the mouse RNA polymerase II CTD, which contains 52 repeats,also appears to affect the efficiency of a function that is essential for cell viability ( 1 05). The effects of CTD deletion mutations were assayed by measuring the ability of mutant genes containing an a-amanitin resistance mutation to confer a-amanitin resistance on transfected rodent cells. RNA polymerase II large subunit genes containing the a-amanitin resistance muta tion and encoding between 36 and 78 heptapeptide repeats were able to confer a-amanitin resistance on transfected cells while those encoding fewer than 25 repeats were not. An intermediate effect was observed when the polymerase gene encoded 29, 3 1 , or 32 repeats; the number and size of transfected a-amanitin-resistant cells were reduced. Like the yeast enzyme, mouse RNA polymerase II is partially defective when the CTD is reduced to approximately half its normal size. The RNA polymerase II CTD has also been shown to be essential in Drosophila. A P-element insertion that truncates the Drosophila CTD from 42 to 20 repeats is lethal when the allele is in the homozygous state ( 1 06). The CTD appears to be a functionally redundant structure whose normal size is important for functional efficiency. Although a considerable portion of the CTD can be deleted in yeast and in mammalian cells without gross alterations in cell viability, the elimination of smaller portions of the CTD can have subtle but important effects on gene expression. For example,the ability to induce INOI expression in yeast is reduced by the loss of only nine heptapeptide repeats (25). It appears that different mammalian cell types may have difft::rent requirements for CTD length or for certain amino acid se quences within specific heptapeptide repeats ( 105). Thus, while portions of the CTD are not required for cell viability in simple growth assays,it seems likely that the entire CTD is necessary for maximal transcriptional efficiency in each eukaryote. Evidence for a Role in Initiation

At most well-characterized promoters, positive and negative regulatory fac tors strike a complex balance to regulate the rate of transcription initiation . Although the role of the RNA polymerase II CTD in transcription is not yet firmly established, the simplest general model that accounts for all of the

708

YOUNG

available data is that the RNA polymerase II CTD is one of several com

ponents of the transcription apparatus that contribute to the response to activating signals from factors associated with the enhancer. The contribution of the CTD to the response to activating signals relative to the contribution of

other mechanisms involved in activation varies at different promoters. GENETIC STUDIES

Yeast cells containing RNA polymerase n with a series

of CTD partial deletion mutations have been constructed to study the effect of


CTD truncation on the ability to transcribe specific genes (25 , 1 03) . RNA

polymerase II CTD mutations can affect the ability of the enzyme to respond to regulatory signals from Upstream Activating Sequences (UASs). Cells containing RNA polymerase II with a large subunit CTD of

1 1 317 repeats are

highly defective in derepression of INO 1 mRNA synthesis, partially defective in derepression of GALl O mRNA synthesis, and unaffected in derepression of HIS4 transcription. The relative ability of an RNA polymerase I I CTD mutant to transcribe

IN01, GALlO, and

HIS4 upon induction correlates with its

relative ability to utilize the UAS elements of these three genes when they

substitute for the CYC1 VAS in vivo. Investigation of the effect of various repeat lengths on utilization of the

INO] , GALlO, and HIS4 UASs confirms

that different UASs vary in their sensitivity to CTD repeat length, and demonstrates that there is a rapid tran sition between repeat lengths that are responsive to VAS-specific activ ati n g signals and those that are not. In adequate

1N01 VAS utilization appears to be a direct result of RNA

polymerase II CTD dysfunction at the INO] promoter, rather than an indirect

result of a lack of INO] transcription factors . Finally, inadequate responses to the GAL4-binding site VAS can account for the inadequate response of CTD mutants to the GALlO UAS. Although the level of GAL4 mRNA is reduced in CTD mutant cells, restoration of GAlA mRNA to wild-type levels does not diminish the defect in the response to the GALlO VAS in CTD mutants. These results implicate the CTD in the response to activation signals from the UAS elements of certain genes.

The inability of RNA polymerase II CTD truncation mutants to respond fully to signals from some transactivating proteins could be a consequence of

several direct or indirect effects. The CTD mutations might affect the ability of the CTD to interact directly with some transactivation factors or their accessory factors . CTD truncation could affect the ability of RNA polymerase to interact appropriately with general transcription factors, which themselves may respond directly or indirectly to transactivation factors . CTD truncation

could alter the conformation of other portions of RNA polymerase, which are normally important for the enzyme's response to transactivating signals. Support for the notion that the RNA polymerase CTD m ay help mediate the response to signals from transcription factors also comes from investigating


RNA POLYMERASE II

709

the effect of RNA polymerase IT with substantially different CTD sizes on transcription from a GALl promoter (the GALl and GALJO promoters share the GALI--JO VAS) in the presence of a variety of GAlA transcriptional activation domain mutations (24). In the GAlA mutants, increases in transcription from the GAL l promoter were observed as the RNA polymerase IT CTD length increased from 1 3 to 38 repeats. In contrast, there was no difference in the ability of RNA polymerase IT with CTD lengths of 1 3, 26, and 38 repeats to express ,B-galactosidase in the presence of wild-type GAL4. The observation that 13 repeats is close to the threshold number of repeats necessary for wild-type responses to the GALl-JO VAS in the presence of wild-type GAL4 (25) may explain why no substantial differences in GALl-JO VAS responses were observed with CTD lengths of 1 3 , 26, and 38 repeats (24). The removal of negative regulators can partially restore the ability of yeast RNA polymerase II with short CTDs to respond to activation signals at specific promoters . The negative regulatory factor SPT2 (also called SIN l ) represses transcription of the yeast HO and INOI genes ( 1 36), among others ( 137). Deletion of the SP12 gene partially restores the ability of RNA polymerasf: II with a CTD of 10 heptapeptide repeats to respond to induction at the INO} promoter, and reduces the severity of the cold-sensitive pheno type of ceilis containing the RNA polymerase mutation ( 1 36). Since CTD truncation severely reduces the ability of RNA polymerase IT to respond to induction at promoters regulated by SPT2 (HO and INOI), and because the absence of SPT2 partially restores the response, one of the roles of the CTD may be to antagonize the negative regulatory activity of SPT2 . It is not yet clear whether the CTD and SPT2 act through the same molecular mechanism. Evidence that the CTD and SPT2 both bind to DNA nonspecifically in vitro led Peterson et al ( 1 36) to suggest that the CTD and SPT2 may compete for DNA binding. It is also possible that the antagonism is more indirect; for example, if the CTD facilitates interaction between upstream transcription factors and the TATA-associated initiation complex, nonspecific DNA binding by SPT2 could curtail that interaction by restricting DNA looping . The results of in vitro transcription experiments BIOCHEMICAL STUDIES thus far indicate that the CTD is required for efficient promoter-dependent transcription at some promoters but not others, and that the CTD is not involved in RNA chain elongation in the presence of naked DNA templates. Monoclonal antibodies directed against the CTD inhibit promoter-dependent transcription from the adenovirus-2 major late promoter and the murine dihydrofolate reductase promoter in HeLa cell nuclear extracts, but do not appear to affect elongation (52 , 1 38, 1 39)_ In addition, synthetic peptides containing the consensus repeat sequence and conjugated to bovine serum


710

YOUNG

albumin (BSA) inhibit specific transcription initiation but not elongation in vitro (52) . Purified calf thymus RNA polymerase II in which the CTD has been removed by proteolysis is not capable of initiation at the murine di hydrofolate reductase promoter (52) . However, purified calf thymus RNA polymerase II lacking the CTD can initiate transcription from the adenovirus2 major late promoter (52, 1 39 , 140). Similarly, purified Drosophila RNA polymerase II lacking the CTD is able to initiate promoter-dependent transcription of Drosophila actin 5C and histone H3 and H4 templates ( 1 06) . Together, these data suggest that the importance of the CTD in transcription initiation varies from one promoter to another, consistent with studies of the ability of RNA polymerase CTD mutants to respond to signals from various UAS elements in vivo (25). In addition, these in vitro transcription ex periments argue against a general role for the CTD in RNA chain elongation. Nuclear extracts of yeast RNA polymerase II CTD truncation mutants are fully competent in promoter-independent initiation and elongation assays and contain normal levels of RNA polymerase II, but are strikingly defective in their response to GAL4-VP I6 stimulated, promoter-dependent transcription in vitro (S . -M . Liao, R . A. Young, unpublished) . Progressive CTD trunca tion results in a progressive loss of GAL4-VP 1 6 stimulated promoter dependent transcription using a template containing a GAL4-binding site UAS . Promoter-dependent transcription can be restored by adding purified wild-type RNA polymerase II to the CTD mutant nuclear extracts, indicating that the defect is not due to the absence of necessary transcription factors . These results confirm that the CTD is important for normal responses to some upstream activating factors . At two promoters where RNA polymerase II lacking the CTD can initiate transcription in vitro, the CTD-less enzyme can respond to stimulation by at least two transcription factors . The calf thymus RNA polymerase CTD is not required to obtain three- to fourfold stimulation of transcription from the adenovirus-2 major late promoter in vitro with the adenovirus Major Late Transcription Factor ( 1 40). In addition . the factor Spl stimulates transcription threefold from a hybrid HSP70 promoter in a Drosophila extract in vitro independent of the presence or the absence of the CTD ( 1 4 1 ) . These results suggest that transcription activation by these factors does not involve the CTD, but it is also possible that these assays do not detect contributions that are normally made by this domain during initiation in vivo. Loss of the CTD does not appear to affect the ability of RNA polymerase II to assemble with general transcription factors on TATA-containing promoter DNA ( 1 40) . When the CTD is removed from mammalian RNA polymerase II by proteolysis, the CTD-Iess RNA polymerase and wild-type RNA polymerase are indistinguishable in their ability to assemble with TATA associated factors on adenovirus-2 major late promoter DNA.

RNA POLYMERASE II

711


General Models for CTD Function

Few aspects of transcription have been left untouched in models that have been proposed for CTD function. These proposed functions include involve ment in transcription initiation, enzyme localization, DNA binding, removal of chromatin proteins from DNA, and general regulation of enzyme activity (55-57, 82, 1 03-1 05 , 1 36, 1 38 , 142, 1 43). The simplest interpretation of all of the available data is that the RNA polymerase II CTD is one of several components of the transcription apparatus that contributes to the response to activating signals from the enhancer. The importance of this contribution relative to the contribution of othcr mechanisms involved in activation varies at different promoters. It seems likely that the CTD acts to facilitate interactions between upstream activating factors (either directly or through additional factors) and com ponents of the mRNA transcription complex (the general transcription factors and/or RNA polymerase) . For example, the hydroxyl-rich CTD could provide an initial target for an upstream transcription factor whose ultimate target is a component of the mRNA transcription complex. Transient weak interactions between heptapeptide repeat sequences and the upstream factor might in crease the rate with which the factor finds the ultimate target in the mRNA transcription complex. Phosphorylation of the RNA polymerase II CTD may play a role in transcription initiation, providing a switch between steps during initiation ( 1 29, 1 3 3 , 144; reviewed in 1 45). The relatively unphosphorylated form of RNA polymerase (IIa) becomes phosphorylated during initiation in vitro. It is difficult to distinguish between a requirement for phosphorylation and coin cidental phosphorylation during this initiation reaction in vitro, but this is a very interesting model. The postulated interaction between the acidic activat ing domain of a transcription factor and the hydroxyl-rich CTD ( 1 43) could be disrupted by phosphorylation of the CTD, permitting a transition between initiation and elongation. If the CTD interacts with DNA in vivo ( 1 36 , 1 42) , that interaction would be disrupted by CTD phosphorylation, permitting a similar typl� of transition. ACTIVArION OF RNA POLYMERASE II The mechanisms by which factors deliver signals to initiate transcription to RNA polymerase II are not yet clear. Signals could be delivered directly through interactions between upstream factors and RNA polymerase, or indirectly through intermediates such as the TATA-associated transcription factors or other cofactors. Whatever the mechanism(s) , RNA polymerase itself must have the capacity to respond to activation signals. The large


712

YOUNG

subunit CTD is one component of RNA polymerase IT that appears to be involved in the response to activating signals. Genetic evidence suggests that other portions of the enzyme may also be involved in this response. For example, an intragenic suppresor of partial CTD truncation mutations occurs within segment H of RPBl ( 1 35), suggesting that this portion of the large subunit interacts with the CTD or is involved in the same function as the CTD. In addition, some mutations in RPB2 affect the transcription of some genes but not others in vivo, and exhibit some but not all of the phenotypes associated with CTD truncations (59). In this context, it is interesting that the E. coli RNA polymerase homologue of RPB2 (f3) probably interacts with the (J subunit; the f3 and (J subunits can be crosslinked to DNA sequences only five nucleotides apart in the lac UV5 promoter ( 1 46) , and mutations in f3 can affect (J binding to the core enzyme (147). How signal transduction to RNA polymerase occurs remains one of the more interesting unknown mechanisms in the control of transcription. CONCLUDING REMARKS A well-defined RNA polymerase IT enzyme provides an important starting point for genetic and biochemical experiments that should improve our un derstanding of transcription and its regulation. Conditional mutations in many of the yeast RNA polymerase IT subunit genes have been isolated (59, 68 , 69, 1 1 2a, 1 48-150) and are being used in combination with in vitro transcription ( 1 5 1) and other biochemical assays to obtain clues to subunit functions. This potent combination of genetics and biochemistry will surely yield valuable new insights into the molecular mechanisms that govern transcription. ACKNOWLEDGMENTS I am especially grateful to Richard Burgess, Steve Buratowski, David Chao, Gerry Fink, Peter Kolodziej , Charles Scafe, and Nancy Woychik for insights, criticism, and stimulating discussions. I thank the many investigators who shared with me their data and thoughts , induding David Baltimore, Ekkehard Bautz, Jean-Marie Buhler, Richard Burgess, Steve Buratowski , Pierre Cham bon, Jeffry Corden , A. Cornelissen, Michael Dahmus, Aled Edwards, Amo Greenleaf, Leonard Guarente, Ira Herskowitz, James Ingles, Carl Mann, Mark Johnston, Roger Kornberg, Mark Mortin, Masayasu Nomura, Craig Peterson, Michel Riva, Alan Sachs, Andre Sentenac, Phil Sharp, Robert Tjian, and Sherman Weissman. I am also grateful to Carolyn C. Carpenter for preparing the manuscript and to David Rothstein for assistance with literature research. This work was supported by Public Health Service Grant GM34365 and by a Burroughs Wellcome Molecular Parasitology Award.

RNA POLYMERASE II

713

Literature Cited M. J. 1 976. RNA Polymerase, ed. R . Losick, M . Cham

I. Chamberlin,

2. 3.


4. 5. 6. 7. 8. 9.

1 0.

berlin, pp. 1 7-67. Cold Spring Harbor, NY: Cold Spring Harbor Lab. 899 pp. Yura, T. , Ishihama, A. 1 979. Annu. Rev. Gmet. 1 3:59-97 Cham�rlin, M . J . 1 982. Enzymes 15: 6 1 -86 McClun!, W. R. 1 985. Annu. Rev. Biochenz. 54: 1 7 1 -·204 Schleif, R. 1 988. Science 24 1 : 1 182-87 Gralla, J. D. 1 989. Cell 57: 1 93--95 Heimann, J . D . , Chamberlin, M . J . 1 988. Annu. Rev. Biochem. 57:839-72 Platt, T. 1 986. Annu. Rev. Biochem. 55:339-72 Bell, S. P. , Learned, R. M . , Jantzen, H . - M . , Tjian, R. 1 988. Science 241 : 1 1 1 92-97 Guarente, L. 1 987. Annu. Rev. Genet.

2 1 :425-52 I I . Johnson, P. F. , McKnight, S. L. 1 989. Annu. Rev. Biochem. 58:799-839 1 2 . Struhl, K. 1 989. Annu. Rev. Biochem.

58: 1 05 1 -77 1 3 . Mitchell , P. J . , Tjian, R. 1 989. Science 245:37 1 -78 1 4 . Guarent'e, L. 1 988. Cell 52:303--5 15. Ptashne., M. 1 988. Nature 335:683-89 1 6 . Geiduschek, E. P. 1 988. Annu. Rev. Biochenz. 57:873--9 1 4 1 7 . V an Dyke, M. W., Roeder, R . G . , Sawadogo, M . 1 988. Science 24 1 : 1 33538 1 8 . Buratowski, S. , Hahn, S . , Guarente, L . , Sharp, P. A. 1 989. Cell 56:549-61 1 9 . Sawadogo, M . , Sentenac, A. 1 990. Annu. Rev. Biochem. 59:7 1 1-54 20. Pugh, B . F ., Tjian, R . 1 990. Cell 6 1 : 1 1 87-97 2 1 . Berger, S. L . , Cress, W. D. , Cress, A . , Triezenberg, S. J . , Guarente, L. 1 990. Cell 6 1 : 1 199-208 22. Kelleher, R . J . , Flanagan, P. M . , Korn berg, R . D. 1 990. Cell 6 1 : 1 209-15 23. Stringer, K. F. , Ingles, C. J., Green blatt, J. 1 990. Nature 345:783--86 24. Allison, L. A . , Inglcs, C. J. 1 989. Proc. Natl. Acad. Sci. USA 86:2794-98 25. Scafe, C., Chao, D . , Lopes, J . , Hirsch, J. P . , Henry, S . , Young, R. A. 1 990. Nature 347:491-94 26. Lewin, B. 1 990. Cell 6 1 : 1 1 6 1-64 27. Ptashne M . , Gann, A. A. F. 1 990. Na ture 346:329-3 1 28. Chambon, P. 1 975. Annu. Rev. Biochem. 44:61 3--38 29. Roeder, R. G. 1 976. See Ref. 1 , pp. 285-329 •.

30. Paule, M . R. 1 98 1 . Trends Biochem. Sci. 6: 1 28--3 1 3 1 . Lewis, M. K . , Burgess, R. R. 1 982. Enzymes 15: 1 09-53 32. Sentenac, A . 1 985. Crit. Rev. Biochem. 1 8 : 3 1 -9 1 33. Roeder, R . G . , Rutter, W . J . 1 969. Na ture 224:234 34. Roeder, R. G., Rutter, W. J. 1 970. Proc. Natl. Acad. Sci. USA 65:675 35. Roeder, R. G . , Rutter, W. J . 1 970. Biochemistry 9:2543 36. Armaleo, D., Gross, S. R . 1 985. J. Bioi. Chem. 260: 1 6 1 69-73 37. D'Alessio, J. M . , Perna, P. J . , Paule, M. R. 1 979. J. Bioi. Chem . 254: 1 1 28287 38. Renart, M. F . , Sastre, L . , Diaz, V . , Sebastian, J . 1 985. Mol. Cell. Biochem. 66:21-29 39. Smith, S. S . , Braun, R. 1 98 1 . FEBS Lett. 1 25: 1 07- 1 0 40. Sanford, T. , Prenger, J. P . , Golomb, M . 1 985. J . Bioi. Chem. 260:8064-69 4 1 . Greenleaf, A. L . , Bautz, E. K. F . 1 975. Eur. J. Biochem. 60: 1 69--79 42. Engelke, D. R . , Shastry, B. S., Roeder, R. G. 1 983. J. Bioi. Chem . 258: 1 92 1 31 43. Schwartz, L . B . , Roeder, R . G . 1 975. J . Bioi. Chem. 250:3221-28 44. Kim, W-Y., Dahmus, M . 1 988 . J. BioI. Chern. 263 : 1 8880-85 45. Freund, E. , McGuire, P. M. 1 986. J. Cell. Physiol. 1 27:432-38 46. Jendrisak, J. J., Petranyi, P. W . , Burgess, R. 1976. See Ref. I, pp. 77992 47. Hodo, H. G. III, Blatti, S. P. 1 977. Biochemistrv 1 6:2334-43 48. Jendrisak, i., Guilfoyle, T. J. 1 978. Biochemistry 17: 1 322-27 49. Guilfoyle, T. J . , Hagen, G., Malcolm, S. 1 984. J. Bioi. Chem. 259:64048 50. Guilfoyle, T. 1. 1 980. Biochemistry 1 9: 5966-72 51 . Kim, W. Y., Dahmus, M. E. 1 986. J. Bioi. Chern. 26 1 : 1 42 1 9-25 52. Thompson, N. E., Steinberg, T. H . , Aronson, D. B . , Burgess, R . R . 1989. J. Bioi. Chem . 264: 1 151 1-20 53. Edwards, A. M . , Darst, S. A . , Feaver, W. J . , Thompson, N. E . , Burgess, R . R . , Kornberg, R . D. 1 990. Proc. Natl. Acad. Sci. USA 87:21 22-26 54. Kolodziej , P. A . , Woychik, N. A . , Liao, S.-M., Young, R . A . 1 990. Mol. Cell. Bioi. 1 0: 1 9 1 5-20


714

YOUNG

5 5 . Allison, L. A . , Moyle, M . , Shales, M . , Ingles, C . J . 1 985. Cell 42:599--6 10 56. Corden, 1 . L., Cadena, D . L., Ahearn, 1. M . , Dahmus, M. E. 1985. Proc. Natl. Acad. Sci. USA 82:7934-38 57. Corden, 1. L. 1990. Trends. Biochem. Sci. 15:383-87 5 8 . Arndt, K . , Styles, c . , Fink, G. R. 1989. Cell 56:527-37 59. Scafe, C . , Nonet, M . , Young, R. A. 1990. Mol. Cell. Bioi. 10: 10 10-- 1 6 6 0 . Kadesch, T. R . , Chamberlin, M . J . 1982. J. Bioi. Chem. 257:5286--95 6 1 . S luder, A. E . , Price, D. H . , Greenleaf, A . L. 1 987. J. BioI. Chern. 263:991725 62. Coulter, D. E. , Greenleaf, A. L. 1985 . J. Bioi. Chern. 260: 13 190--98 63 . Proudfoot, N. J. 1989. Trends Biochem. Sci. 14: 1 05-10 64. Young, R. A . , Davis, R. W. 1983. Sci ence 222:778-82 65. Ingles, C. J . , Himmelfarb, H. J . , Shales, M . , Greenleaf, A . L . , Friesen, J. D. 1984. Proc. Natl. A cad. Sci. USA

8 1:2 157-61 66. Riva. M Memet. S . , Micouin. J . Y . , Huet, J . , Treich, I . , et al. 1986. Proc . Natl. Acad. Sci. USA 83: 1 5 54-58 67. Sweetser, D . , Nonet, M . , Young, R. A . 1987 . Proc. Nat!. Acad. Sci. USA 84: 1 1 92-96 68. Kolodziej, P. A . , Young, R. A. 1989. Mol. Cell Bioi. 9:5387-94 69. Woychik, N . A . , Young, R . A. 1989. Mol. Cell BioI. 9:2854-59 70. Woyehik, N. A . , Liao, S . -M . , Kolod ziej, P . , Young, R. A. 1 990. Genes Dev. 4: 3 1 3-2 3 7 1 . Woyehik, N. A . , Young, R. A. 1990. J. Biol. Chem. 265 : 178 1 6-- 19 72. Woychik, N. A . , Lane, W. S . , Young, .•

R. A. Submitted

73. Searles, L. L . , Jokerst. R. S . , B ingham. P . M . , Voelker, R. A. , Greenleaf, A. L. 1982. Cell 3 1:585-92 74. Breant, B . , Huet, J . , Sentenae, A . , Fro mageot, P. 1983. J. Bioi. Chern. 258 : \ 1968-73 7 5 . Buhler, J . -M . , Huet, J . , Davies, K. E . , Sentenac, A . , Fromageot, P . 1980. J. BioI. Chern. 255:9949-54 76. Jokerst, R. S . , Weeks, J. R . , Zehring , W. A . , Greenleaf, A. L. 1 989. Mol. Gen. Genet. 2 1 5:266--7 5 77. Cornelissen , A . W . C. A . , Evers, R . , Kock, J . 1988. Oxford Surv. Eukaryotic Genes 5:91-13 1 78. Memet, S . , Gouy, M . , Marek, C . , Sen tenac, A . , Buhler, J . -M . 1988. J. Biol. Chern . 262:2830--3 9 79. Huet, J . , Sentenac. A .. Fromageot, P. 1982. J. BioI. Chern. 257:2613-18

80. Weeks, J . R. , Coulter, D. E . , Green leaf, A. L. 1982. 1. Bioi. Chern. 257:5884-91 8 1. Bird. D. M . . Riddle. D. L. 1989. Mol. Cell. Bioi. 9:4119-30 82. Ahearn, J. M. Jr. . Bartolomei. M. S . . West, M . L . , Cisek, L . J . , Corden, J . L. 1987. J. Bioi. Chern. 262 : 10695-705 83. Evers, R . , Hammer, A . , Kock, 1 . , Jess, W . , Borst, P . , et al. 1989. Cell 56:58597 84. Smith, J. L . , Levin, 1 . R . , Ingles, C. J . , Agabian, N . 1989. Cell 56:8 15-27 85. Falkenburg, D . , Dworniczak, B . , Faust, D . , Bautz, E. K. F. 1987. J. Mol. BioI. 195:929-37 86. Li , W.-B . , Bzik, D. J . , Gu, H . , Tanaka, M . , Fox, B . A . , Inselburg, J. 1989. Nucleic Acids Res. 1 7 : 962 1 -36 87. Zillig , W. , Palm, P . , Heil, A. 1 976. See Ref. 1, pp. 10 1-25 88. Gundelfinger, E. D. 1983. FEBS Lett. 1 57 : 1 33-38 89. Horikoshi , M . , Tamura, H. 0 . , Sekimi zu, K . , Obinata, M . , Natori , S. 1 983. J. Biochern. 94: 176 1-67 90. Chuang, R. Y . , Chuang, L. F. 1987. Biochern. Biophys. Res. Cornrnun. 1 45 : 73-80 9 1 . Carroll, S . B . , Stollar, B . D. 1983. J. Mol. Bioi. 1 70:717-90 92. Bartholomew, B . , Dahmus, M. E . , Meares, C . F. 1986. J . Biol. Chern.

26 1 : 14226--3 1 93 . Hanna, M . M . , Meares, C. F. 1 983. Proc. Natl. Acad. Sci. USA 80:4238-42 94. Graehev, M . A . , Hartmann, G. R . , Maximova, T. G . , Mustaev, A . A . , Schaffner, A . R . , et al. 1986. FEBS Lett. 200:287-90 95. Grachev, M. A . , Kolocheva, T . , Lukh tanov, E. A . , Mustaev, A. A. 1987. Eur. I Biochern. 1 63 : 1 13-21 96. Riva, M . , Schaffner, A. , Sentenac, A . , Hartmann , G . , Mustaev, A . , e t a l . 1 987. J. BioI. Chern. 262: 14377-80 97. Grachev, M . A . , Lukhtanov, E. A . , Mustaev, A . A . , Zaychikov, E. F . , Abdukayumov, M . N . , et al. 1989. Eur. J. Biochern. 1 80:577-85 98. Berghofer, B . , Krockel , L . , Kortner, .

.

C . , Truss, M . , Schallenberg, J . , Klein, A. 1988. Nucleic Acids Res. 16: 8 1 1 3-28 99. Riva, M . , Carles, C . , Sentenac, A . , Grachev, M . A. , Mustaev, A. A . , Zayehikov, E. F . 1990. J . BioI. Chern.

265: 16498-503 1 00. Ovchinnikov, Y . , Lipkin, V. M . , Mod

yanov, N. N . , Chertov, O. Y . , Smirnov, Y. V. 1977. FEBS Lett. 76: 108- 1 1 1 0 1 . Kolodziej , P . 1 99 1 . PhD thesis, MIT 102. Pati, U. K . , Weissman, S. M. 1990. 1.

BioI. Chern. 265:8400--3

RNA POLYMERASE II 1 0 3 . Nonet, M . , Sweetser, D . , Young, R. A . 1 987. Cell 50:909- \ 5 1 04. Allison, L. A . , Wong, J . K . -C . , Fitzpat rick, V . D . , Moyle, M . , Ingles, C. J. 1988. Mol. Cell. Bioi. 8:321-29 1 0 5 . Bartolomei, M. S . , Halden, N. F . , Cul len, C. R. , Corden, J. L. 1 988. Mol. Cell. Bioi. 8:330--39 1 06 . Zehring , W. A . , Lee, J. M . , Weeks, J . R . , Jokerst, R . S . , Greenleaf, A . L . 1 98 8 . hoc. Nat!. Acad. Sci. USA


85:3698-702

1 07 . Cadena, D . , Dahmus, M. 1987 . J. Bioi. Chern. 262: 1 2468-74

1 0 8 . Buhler, J.-M. , Iborra, F., Sentenac, A . , Fromagt:ot, P . 1976. FEBS Lett. 7 1 :3741 109. Buhler, J. -M . , Iborra, F., Sentenac, A . , Fromageot, P. 1 976. J . Bioi. Chern. 25 1 : 1 7 1 2- 1 7 1 10 . Valenzuela, P . , Bell, G . 1. , Weinburg, F . , Rutter, W. J. 1 976. Biochern. Bio phys. Res. Cornrnun. 7 1 : 1 3 1 9-25 1 1 1 . Huet, J . , Phalente, L. , Buttin, G . , Sen tenac,

A . , Fromageot, P. 1 982. EMBO

J. 1 : 1 1 9 3-98 1 1 2 . Huet, J . , Riva, M . , Sentenac , A . , Fro mageot, P. 1985. J. Bioi. Chern. 260:

1 5304--1 0 1 1 2a . Archambault, J . , Schappert, K . T . , Friesen, J . D . 1990. Mol. Cell. Bioi.

10:6123--3 1 1 1 3 . Dezelee . S . , Wyers, F . , Sentenac, A . , Fromag(:ot, P. 1 976. Eur. J . Biochern. 65:543-52

1 14. Ruet, A . , Sentenac, A . , Fromageot, P . , Wi n sor, B . , Lacrou te , F. 1 980. 1. Bioi. Chern. 255 :6450--55

1 1 5 . Edwards , A. M . , Kane, C. M . , Young, R. A . , Kornberg , R. D. 1 99 1 . J. Bioi. Chern . 266 : 7 1 -7 5 1 16 . Solaiman, D . , Wu, F. Y . -H . 1984. Biochernistry 23:6369-77 1 1 7 . Lattke, H . , Weser, U. 1976. FEBS Lett.

65:288-91

1 1 8 . Martin, C, Okamura, S . , Young, R. A. 1990. Mol. Cell. Bioi. 10: 1 908- 1 4 1 1 9. Mortin, M . A . 1 990. Proc. Natl. Acad.

Sci. USA 87:4864--68

1 20. Yano, R . , Nomura, M. 1 99 1 . Mol. Cell. Bioi. 1 1 :754--64

1 2 1 . Darst, S . , Edwards, A . , Kuba1ek, E. ,

Kornberg, R. 1 990. Submitted 1 22 . Darst, S . A . , Kubalek, E. W . , Korn berg, R. D. 1989. Nature 340:730--32 1 23 . Faust, D. M . , Rcnkawitz-Pohl, R . , Fal kenburg . D . , Gasch, A . , Bailojan, S . , et al. 1 986. EMBO J. 5:74 1-46 1 24 . Pati U. K. , Weissman, S. M. 1 989. J. ,

Bioi. Chern. 264: 1 3 1 14--2 1

1 25 . Parkhurst, S . M . , Harrison, D . A . , Remington, M . P. , Spana, C . , Kelly, R . L . , e t al . 1 988. Genes Dev. 2 : 1 205- 1 5 1 26. Puhler, G. , Leffers, H . , Gropp, F . ,

715

Palm, P . , Klenk, H . -P . , et al. 1989. Proc. Natl. Acad. Sci. USA 86:4569-73

1 27 . Ovchinikov, Y. A . , Monastyrskaya, G . S . , Gubanov, V . V . , Gu rye v S . 0 . , Salomatina, I . S . , e t a l . 1982. Nucleic Acids Res. 10:4035-44 1 28 . Broyles , S. S . , Moss, B. 1 986. Proc. Natl. Acad. Sci. USA 83 : 3 1 4 1-45 1 28a. Dietrich, M. A . , Prenger, J. P . , Guil foyle, T. J. 1 990. Plant Mol. BioI. 1 5 : 207-23 1 29. Payne, J. M . , Laybourn P. J . , Dahmus, M. E. 1989. J. Bioi. Chern. 264 : 1 962129 1 30. Cisek, L . , Corden, J . 1 989. Nature ,

,

339:679-84 J . M . , Greenleaf, A . L . 1 989. Proc. Natl. Acad. Sci. USA 86:3624--28 Evers, R. , Hammer, A . , Cornelissen, A. W. C. A. 1989. Nucleic Acids Res. 17:3404--13 Laybourn. P. J . Dahmus , M. E. 1 989. J. Bioi. Chern. 264:6693-98 Bell, G. 1 . , V alenzuela, P . , Rutter, W , J . 1977. J. Bioi. Chern. 252:3082-9 1 Nonet, M . , Young, R. A. 1 989. Genet ics 1 23 : 7 1 5-24 Peterson, c . , Kruger, W . , Herskowitz, 1. 1 99 1 . Cell. In press Roeder, G. S . , Beard, c. , Smith, M . , Keranen, S . 1 985. Mol. Cell. Bioi. 5 :

1 3 1 . Lee,

132. 133. 1 34 . 135.

1 36. 137.

.

1 543-53 138. Moyle, M . , Lee, J. S . , Anderson , W . F . , Ingles, C . J . 1 989. Mol. Cell. Bioi. 9:5750--53 1 39. Kim, W . -Y . , Dahmus, M. E. 1989. J.

Bioi. Chern. 264:3 1 69-76

140. Buratowski, S . , Sharp, P. 1990. Mol. Cell. Bioi. 10:5562-64 1 4 1 . Zehring, W. A . , Greenleaf, A . L . 1990.

J. BioI. Chern. 265:8351-53 1 42 . Suzuki, M . 1 990. Nature 344:562-65 143. Sigler, P. 1 988. Nature 333 : 2 1 0-- 1 2 144. Laybourn, P. J . , Dahmus, M . E. 1 990. 1. Bioi. Chern. 265 : 1 3 1 65-73 145. Dahmus, M . E . , Dynan , W. S . 1 991 . Transcriptional Regulation, ed. K . Yamamoto, R . Tjian. Cold Spring Har bor, NY: Cold Spring Harbor Lab. In press 146. Simpson, R . B. 1 979. Cell 1 8 : 277-85 147 . Glass, R. E . , Honda, A . , Ishihama, A . 1 986. Mol. Gen. Genet. 203 : 4 92-95 148. Scafe, C . , Martin, c . , Nonet, M . , Podos, 5 . , Okamura, S . , Young, R . A .

1990. Mol. Cell. Bioi. 1 0 : 1 270--7 5 1 49. Nonet, M . , Scafe, c . , Sexton, J . , Young, R . A . 1987. Mol. Cell. Bioi. 7 : 1 60 1- 1 1

1 50. Himmc1farb, H. J . , Simpson, E. M . , Friesen, J . D . 1 987. Mol. Cell. Bioi. 7:21 55-64

1 5 1 . Lue, N. F. , Kornberg , R. D. 1987. Proc. Natl. Acad. Sci. USA 84:8839-43

Dephosphorylating eukaryotic RNA polymerase II.

[Focus on RNA polymerase II].

Tails of RNA polymerase II.

A bumpy road for RNA polymerase II.

Advances in RNA polymerase II transcription.

Interchangeable RNA polymerase I and II enhancers.

Phaseolinone, a new mycotoxin, inhibits RNA polymerase(s) other than RNA polymerase II.

RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro.

Strand-specific, high-resolution mapping of modified RNA polymerase II.

Chromatin modification by the RNA Polymerase II elongation complex.

UV damage-induced RNA polymerase II stalling stimulates H2B deubiquitylation.

RNA polymerase II depletion promotes transcription of alternative mRNA species.

Understanding the Molecular Basis of RNA Polymerase II Transcription.

Imaging RNA Polymerase II transcription sites in living cells.

Rat1p maintains RNA polymerase II CTD phosphorylation balance.

Transcription: splicing keeps RNA polymerase II in check.

Roles of TFIID in transcriptional initiation by RNA polymerase II.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

Stability of Drosophila RNA polymerase II elongation complexes in vitro.

Characterization of RNA polymerase II-dependent transcription in Xenopus extracts.

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression.

RNA Polymerase II Trigger Loop Mobility: INDIRECT EFFECTS OF Rpb9.

Brd4 activates P-TEFb for RNA polymerase II CTD phosphorylation.

Complete sequence of the human RNA polymerase II largest subunit.