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Molecular and Cellular Biology, September 2001, p. 6056-6065, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.6056-6065.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Partners of Rpb8p, a Small Subunit Shared by Yeast
RNA Polymerases I, II, and III
Jean-François
Briand,1
Francisco
Navarro,1,
Peggy
Rematier,1
Claire
Boschiero,1
Sylvie
Labarre,1
Michel
Werner,1
George V.
Shpakovski,1,2 and
Pierre
Thuriaux1,*
Service de Biochimie and
Génétique Moléculaire, CEA/Saclay, F-91191
Gif-sur-Yvette, France,1 and
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, 117871 Moscow GSP-7 V437,
Russia2
Received 27 December 2000/Returned for modification 5 February
2001/Accepted 6 June 2001
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ABSTRACT |
Rpb8p, a subunit common to the three yeast RNA polymerases, is
conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly
conserved amino acids. With one exception, they are clustered on the
Rpb8p structure. They all impair a two-hybrid interaction with a
fragment conserved in the largest subunits of RNA polymerases I
(Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to
the pore 1 module of the RNA polymerase II crystal structure and bears
a highly conserved motif (P.I.KP..LW.GKQ) facing the GGLLM motif of
Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the
invariant glycyl of P.I.KP..LW.GKQ provokes a
temperature-sensitive defect. Increasing the gene dosage of another
common subunit, Rpb6p, suppresses this phenotype. It also suppresses a
conditional growth defect observed when replacing Rpb8p by its human
counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo.
These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not
included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-
null allele. A two-hybrid
interaction also occurs between Rpb8p and the product of orphan gene
YGR089w. A ygr089- null mutant has no
detectable growth defect but aggravates the conditional growth defect
of rpb8 mutants, suggesting that the interaction with
Rpb8p may be physiologically relevant.
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INTRODUCTION |
Three related RNA polymerases
(Pol's) are required to transcribe the nuclear genome of all
eukaryotes investigated so far. In yeast (Saccharomyces
cerevisiae), they are made of 14 (Pol I), 12 (Pol II), and 17 (Pol
III) polypeptides. Ten of these subunits form a conserved core
structure containing the two large polypeptides (corresponding to the
bacterial 
' and ) and eight small subunits with molecular masses
ranging between 8 and 25 kDa (38). The Pol II core
enzyme is competent for the transcription of nonspecific DNA templates
(12). In the cases of Pol I (30) and Pol III (5, 37), it has been demonstrated that additional
enzyme-specific subunits are required for promoter-dependent
transcription. The backbone structure of the yeast core Pol II
(9) reveals a remarkable similarity to the bacterial core
structure (43). Both enzymes contain a large DNA channel,
leading to an internal catalytic pocket containing one or possibly two
catalytic Mg2+ ions. A secondary channel beneath
the active site probably allows nucleotide triphosphates to gain access
to the catalytic site. This catalytic architecture is primarily
determined by the folding of the two largest subunits.
The role of the small polypeptides present in the eukaryotic core
structure is still poorly characterized. Four of them form an 
-like
structure (9, 22). Like the corresponding bacterial 2 homodimer, this structure is located at the
rear of the bacterial enzyme and has been implicated in protein
assembly but does not appear to directly contribute to the active site
(16, 20, 22, 25, 32). It contains two small polypeptides
(Rpb10p and Rpc10p) common to the three nuclear enzymes
(7) and two subunits shared by Pol I and III (Rpc40p,
Rpc19p) which have strong homologs in Pol II (Rpb3p, Rpb11p). It has
been known for a long time that Pol's I, II, and III share three other
small polypeptides, Rpb5p, Rpb6p, and Rpb8p (6, 39, 42).
The first two polypeptides are strongly conserved from archaea to
eukaryotes (23), and Rpb6p is also distantly related to
the bacterial 
subunit (28). The present study deals
with Rpb8p, a 16.5-kDa protein that has no homology in archaeal or
bacterial genomes and is thus a uniquely eukaryotic component of the
transcription machinery.
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MATERIALS AND METHODS |
Genetic material.
Common yeast media, growth conditions, and
genetic techniques were used as described elsewhere (15, 34,
36). Yeast-peptone-dextrose (YPD) is a standard rich
medium, synthetic complete (SC) is a synthetic medium supplemented with
amino acids, adenine, and uracil, and SC-U is the corresponding medium
without uracil. Strains and plasmids are listed and summarily described
in Tables 1 and
2. Gene symbols and their synonyms are
listed in the Yeast Protein Data website
(http://www.proteome.com/databases/index.html). The corresponding
protein symbols are followed by a p (Rpb8p), and the product of the
YGR089w open reading frame was thus noted Ygr089p. The human
homologue of Rpb6p and Rpb8p were referred to as Hs6 and Hs8.
The
ygr089w-::URA3 null mutant was constructed
by deleting an internal 810-bp
HindIII fragment of
YGR089w and replacing it
with a 1,170-bp
URA3+ fragment. The corresponding
SalI-
ClaI fragment was introduced
into a
YPH499 × YPH500 diploid strain by transformation. Integrative
transformants generated by homologous recombination at the
YGR089w locus were selected on SC-U and confirmed by PCR
amplification
from primers upstream and downstream of
YGR089w. The mutant haploid
strains YPR2 and YPR3 were
obtained by meiotic sporulation. pCM189-Hs6
was constructed by cloning
the PCR-amplified human
RPB6 cDNA (Hs6)
in pCM189
(
17). Site-directed mutagenesis of pGEN-RPB8 and
pGBT9-RPB8
was done by PCR amplification with
Pfu DNA
polymerase (Stratagene),
using complementary primers containing the
desired mutation. PCR
products were digested with
DpnI to
eliminate the methylated wild-type
template and propagated in XL1-Blue
Escherichia coli cells. Mutant
clones were identified by DNA
sequencing and checked for the absence
of additional mutations in the
RPB8 coding sequence. Their growth
phenotype was tested by
genetic transformation in strain YGVS043,
using a plasmid shuffle assay
(
34).
Two-hybrid tests were as described before (
13). Starting
from a random library of genomic fragments (
14), 29 clones
were
isolated from 1.2 × 10
7 transformants
by a double selection based on the activation of
the
HIS3
and
LacZ reporter genes. Five clones were duplicate isolates
due to some redundancy in the DNA library, four corresponded to
noncoding regions, three were out-of-frame fusions, and three
were
FAS3 fragments that were isolated in several other screens
of the same library. The 15 remaining clones resulted from in-frame
fusions in eight distinct genes. Dose-dependent suppressors of
the
temperature-sensitive growth defect of strain YGVS045 were
isolated
from a yeast genomic library as previously described
(
36).
Cell fractionation.
Log-phase cells grown in 250 ml of YPD
to an optical density at 600 nm (OD600) of about
0.6 were harvested by centrifugation, washed twice with spheroplasting
buffer (50 mM Tris-HCl [pH 7.5], 1.2 M sorbitol, 10 mM
NaN3, and 40 mM -mercaptoethanol), and resuspended in a final volume of buffer adjusted to 1 ml for 40 OD600 units. Zymolyase 100T (ICN Biomedicals) was
added at 300 µg/ml. Cells were incubated at 37°C for 30 to 45 min,
and spheroplast formation was monitored under a microscope. Cells were
washed twice in the same buffer, resuspended in 1 volume of ice-cold lysis buffer (20 mM HEPES-KOH, pH 7.4, 100 mM K-acetate, 5 mM Mg-acetate, 1 mM EDTA, 1 mM DTT) with protease inhibitors (Boehringer Mannheim) and 1 mM phenylmethylsulfonyl fluoride. They were gently crushed in a cold Potter, centrifuged for 3 min at 1,000 × g to remove unbroken cells, and centrifuged again for 15 min
at 13,000 × g. The pellet (P13) contained nucleus and
some endoplasmic reticulum fragments. The supernatant was purified by
ultracentrifugation at 100,000 × g for 1 h,
yielding an S100 fraction that contained essentially cytosolic
proteins. Twenty to 100 µg of proteins separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was blotted to
nitrocellulose membranes (Hybond C-super) and probed with antibodies
using the ECL detection system (Amersham Pharmacia Biotech).
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RESULTS |
Rpb8p interacts in vivo with Rpb6p.
Figure
1 documents the sequence conservation of
Rpb8p, based on the inspection of current databanks. This subunit is
present in fungus (S. cerevisiae, Schizosaccharomyces
pombe), plant (Arabidopsis thaliana), and animal
(Drosophila melanogaster, Caenorhabditis elegans,
Homo sapiens) genomes. Rpb8p is likely to be shared by all
three RNA polymerases in each of these organisms, since the corresponding gene is unique on their genome. Figure 1 shows that Rpb8p
contains about 40 highly conserved or invariant positions (i.e., less
than one-third of the protein) shared among all the Rpb8p-like subunits
identified so far. A disorganized 
loop (positions 68 to 88)
is totally dispensable in vivo (40) and is absent from
animal or plant Rpb8p. The spatial structure of the yeast subunit was
determined in solution by nuclear magnetic resonance (NMR)
(21) and, more recently, by high-resolution crystal data from the core of Pol II (8). These two structures are in
fairly good agreement but are not entirely superimposable. The most
highly conserved regions on the amino acid sequence correspond to the 1-, 4-, 6-, and 10-strands and to an invariant GGLLM
signature between 8 and 9.
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FIG. 1.
Sequence conservation and site-directed mutagenesis of
Rbp8p. Sc, S. cerevisiae; Sp, S. pombe;
Hs, H. sapiens; Dm, D. melanogaster; Ce,
C. elegans; At, A. thaliana. Shown are
invariant or highly conserved positions (red) and positions conserved
in at least three sequences (blue). The positions of the -strands
identified on the spatial structure (8, 21) are indicated
by horizontal lines. A GGLLM signature motif present in Rpb8p-like
sequences is noted by a red bar. The 17 amino acids mutated in this
study are indicated by triangles; black triangles denote mutations with
no growth phenotype (F6L, F6K, F10D, P17K, P48V, D53A, Y95F, Y95R,
Y95E, M97C, M97D, M97K, Y115F, Y115R, Y115E, L121I, L121N, L121K,
L121D, R124A, N131K) (the rpb8-21
deletion of the -loop is also silent; 40), and red
triangles indicate conditional (L38D, G99D, G99K, L122D) and lethal
(S117D, S117K, G119D, G120K, G120D) mutations.
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The human subunit (Hs8) differs from
S. cerevisiae by 107 of
its 150 amino acids, with up to 80 nonconservative changes.
Accordingly,
replacing the yeast subunit by its human counterpart
(strain YGVS045)
amounts to a major multisite modification. We have
previously
shown that complementation between yeast and human
RPB8 is functional
at 30°C, with a sharp growth defect at
37°C (
34) (Fig.
2A).
Moreover, the wild-type and hybrid Pol I (containing Hs8 instead
of
Rpb8p), purified from cells grown at the permissive temperature,
have
an identical subunit composition (Fig.
2B) with a comparable
specific
activity on a poly(dA-dT) template, indicating that Hs8
is effectively
and stably incorporated in the yeast enzyme under
these conditions
(data not shown).
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FIG. 2.
Genetic interactions between Rpb6p and Rbp8p. (A) Strains
YGVS043 (rpb8-::LYS2 with
the yeast RPB8 gene [WT]), YGVS045
(rpb8-::LYS2 with the human
Hs8 gene [Hs8]), YJB026 (YGVS045 with a centromeric plasmid
expressing the yeast RPB6 gene [Hs8 + Rpb6p]), and
YJB027 (YGVS045 with a centromeric plasmid expressing the human Hs6
gene [Hs8 + Hs6]) were streaked on YPD and incubated for 3 days at
37°C. Strain genotypes are listed in Table 1. (B) Five micrograms of
a highly purified and catalytically active preparation of Pol I and of
two purified preparations from YGVS043 (WT) and YGVS045 (Hs8) strains
was separated by SDS-8 to 12% PAGE and silver stained. Individual
subunits were identified from their apparent molecular weight, in
parallel with standard protein markers (lane M) and a purified Pol I
sample (15). (C) Strains JAY444
(rpb6-::LEU2 with the yeast
RPB6 gene [WT]), YGVS003
(rpb6-::LEU2 with the
human Hs6 gene [Hs6]), YGVS004
(rpb6-::LEU2 with the
human Hs6 gene and an S2A mutation [Hs6-S2A]) were streaked on
YPD and incubated for 3 days at 30°C, showing the same growth pattern
(this observation was made at temperatures ranging from 16°C to
37°C). Strain constructions and genotypes are in Table 1.
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The temperature-sensitive (ts) growth defect associated with the human
Rpb8p subunit (Hs8) provided a simple way to look for
physiological
partners of Rpb8p by dose-dependent extragenic suppression
(
36). Of the 12 clones thus obtained, three contained
RPB8 itself.
The nine others harbored
SSD1 (four
clones),
UBI2 (three clones),
UBI4 (one clone),
and
SMY2 (one clone).
UBI2 and
UBI4
(encoding
ubiquitin) also weakly suppress a temperature-sensitive Pol
III
mutant,
rpc31-
236. Ubiquitin targets proteins
for degradation
by the 26S proteasome, and suppression may therefore be
due to
the proteolysis of incorrectly folded mutant polypeptides.
SMY2 and
SSD1 suppress various mutants unrelated
to Pol's (
24,
36)
and may have a general effect on the
adaptation of
S. cerevisiae at 37°C.
We separately tested all RNA polymerase subunits and found strong
suppression by
RPB6 (encoding the common subunit Rpb6p),
even when it was on a centromeric vector (Fig.
2). Note that
centromeric
vectors have a low copy number, but are not necessarily
single
copy, and that genes may be expressed at higher levels than in
a
chromosomal context (
2). Suppression was specific of the
human allele (Hs8), with no effect on other conditional mutants
(
rpb8-G99D,
rpb8-G99K, and
rpb8-L122D)
obtained by site-directed
mutagenesis (see below).
RPB6 also
strongly suppressed
rpa190-G728D,
a Pol I with a temperature
sensitivity mutation in the largest
subunit (Fig.
3). This mutant, initially called
rpa190-
3 (
41),
corresponds to the
invariant Gly of a highly conserved P.I.KP..LW.GKQ
motif characteristic
of domain e (
38), a conserved region present
on the three
eukaryotic Pol's and on the archaeal enzyme but absent
from bacteria.
On the core Pol II structure, this motif corresponds
to a short
18-
19-
16 fold where the invariant glycine marks
the beginning
of
16 (
8). As outlined below, this motif is
located
across Rpb8p on the spatial structure (Fig.
4) and is
part of a conserved domain that
is specifically recognized by
Rpb8p on the largest subunits of Pol's
I, II, and III (
13).
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FIG. 3.
Suppression of rpa190-G728D by
RPB6. (A) Strain NOY265 (41) was
transformed by URA3+ plasmids bearing
the yeast (2µm RPB6) or human (2µm Hs6) RPB6 genes
or the centromeric URA3 plasmid bearing the yeast
RPB6 gene (CEN RPB6). The control strain was NOY265
transformed with a void plasmid (pFL44L [vector]). Cells were
streaked on YPD and incubated for 5 days at 37°C. (B) A local
alignment between the largest subunits of yeast (Rpa190p, Rpb1p,
Rpc160p), human Rpb1p (Rpb1p Hs), and archaeal (A' Sa) RNA polymerases.
The black triangle indicates the position of the
rpa190-G728D mutation. *, identity; +, similarity.
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FIG. 4.
Spatial organization of Rpb8p and Rpb6p on Pol II. This
figure was prepared with the RASMOL software
(ftp://ftp.dcs.ed.ac.uk/pub /rasmol), using yeast Pol II
coordinates (PDB accession codes, 1I3Q and 1I50; see reference
8). (Top) Front and side views approximately corresponding
to the synomymous views used by Cramer et al. (8, 9).
Rpb8p (green), Rpb6p (blue) without its first 71 amino acids, forming a
disorganized tail (9, 11), and part of Rpb1p (between
positions 508 and 663) are space-filled, the rest of the core Pol II
structure being given as a C backbone, with the 'F bridge domain
(positions 809 to 845 of Rpb1p) shown as an -helix (see references
8 and 9 for more details on the spatial
structure). Orange, two-hybrid interacting domain recognized by Rpb8p
(positions 516 to 639; 13); violet, positions 563 to 580, corresponding to the invariant P.I.KP..LW.GKQ motif (see Fig. 3);
yellow, part of the pore 1 module (positions 508 to 663; see reference
8) that is not included in the two-hybrid interacting
domain; grey, K738 and D974, two positions that closely contact Rpb8p
on the structure but are not included in the two-hybrid fragment. The
catalytic Mg2+ is represented by a red sphere (not drawn to
scale). (Bottom) Scaled-up views of the front and side views shown
above, with slightly different viewing angles. The two-hybrid
interacting domain is shown as an -helical and -stranded ribbon
structure, with the same color code as above. Position G574
(corresponding to the rpa190-G728D mutation of Pol I;
see Fig. 3) is space-filled and shown in black. Some of the Rpb8p
positions mutagenized in this study are individualized in blue
(phenotypically silent mutations) or red (lethal or conditional growth
defects). The dispensable loop (partly disorganized on the Rpb8p
structure) (8, 21) is also indicated.
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Despite their strong physiological interaction, Rpb6p and Rpb8p do not
directly contact each other on the high-resolution
Pol II structure
(
8) (Fig.
4). However, dose-dependent suppression
is
mediated by a mass action effect of the overproduced suppressor
gene
and need not imply a direct protein contact. Moreover, the
relevant
interaction could occur at an early stage of enzyme assembly.
A closer
look at the spatial structure shows that Rpb8p and Rpb6p
are
essentially separated by a module of the largest subunit (positions
508 to 663 on Rpb1p), called pore 1 (
9). Accordingly, the
dose-dependent
suppression effect of
RPB6 on
rpa190-G728D and on the temperature
sensitivity phenotype
associated with Hs8 could be mediated by
an allosteric rearrangement
involving the pore 1
module.
On the other hand, the present Pol II structure lacks the first 71 amino acids of Rpb6p that form a highly disordered and
very acidic
domain in the yeast subunit (
9,
33) and in the
NMR
structure of the human subunit (
11). The N end might
conceivably
protrude out of the Pol II structure to reach Rpb8p,
notwithstanding
the fact that the 72nd amino acid of Rpb6p (K72) is
located away
from Rpb8p on the Pol II structure. The human Rpb6p (Hs6),
which
essentially differs from the yeast Rpb6p by its shorter acidic
N
tail, complements an
S. cerevisiae null mutant (
26,
34).
It has no suppressor effect on the temperature sensitivity
phenotype
of the human Rpb8p (Hs8) when borne on a centromeric plasmid
(Fig.
2A, Hs8 + Hs6) and only weakly suppresses when expressed from
a
multicopy plasmid (data not shown). The yeast and human Rpb6p
are
phosphorylated (
3), and this phosphorylation occurs at
the
N-terminal Ser2 position of the human subunit (
19).
However,
a S2A replacement at this position effectively complements the
yeast
rpb6-
null mutant (Fig.
2C), indicating that
phosphorylation
is not strictly critical for growth, although it may
have some
subtle role in
vivo.
Rpb8p interacts with conserved domain e on largest subunit of
Pol's I, II, and III.
To search for more putative partners of
Rpb8p, we used a systematic two-hybrid screening, based on a yeast
genomic library containing random fusions to the Gal4p activation
domain (14). In this approach, a pGBT9-RPB8 fusion of
Rpb8p to the Gal4p DNA binding domain was used as bait, and it yielded
15 independent positive clones (see Materials and Methods and Fig.
5). Five of them encoded homologous
segments of the largest subunit of Pol's II (Rpb1p, one clone) and III
(Rpc160p, four clones), and an ad hoc construction bearing the
homologous domain of the Pol I subunit Rpa190p also responds to
pGBT9-RPB8 (13). Four other clones corresponded to
overlapping fragments of the orphan gene YGR089w, which may
thus conceivably encode a physiological partner of Rpb8p (see below).
NUP82, encoding a yeast nucleoporin (18), was
represented by two isolates. The remaining six clones were each
represented by a different gene (GCR1, PET309,
UBP14, and CUP1) and, given their known
physiological role or extranuclear localization, almost certainly have
no physiological interaction with Rpb8p.
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FIG. 5.
Two-hybrid interactions of Rpb8p. (A) Effect of Rpb8
mutations on growth and two-hybrid interactions. Growth patterns were
determined on YPD plates streaked and incubated for 3 to 5 days at 25, 30, and 37°C. cs, little or no growth at 25°C; ts, little or no
growth at 37°C. Strengths of the two-hybrid interactions are as
follows: ++, wild-type level of interaction; +, partly defective
interaction; ( ), weak interaction; , no interaction. The plasmids
bearing rpb8 mutant baits were all derived from
pGBT9-RBP8 by site-directed mutagenesis. The prey plasmids were
pACT2-RPA190e, pACT2-RPB1e, pACT2-RPC160e, pACT2-NUP82, and
pACT2-YGR089w (see Table 1). (B) -Galactosidase plate assays of
two-hybrid interactions. The assay is illustrated by the wild-type,
human, and S. pombe RPB8 and rpb8-L122D
alleles. The negative control corresponds to the void pGBT9 plasmid
(13).
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The Rpb1p fragment recognized by Rpb8p in this two-hybrid assay is
comprised of amino acids 516 to 639. The N-terminal half
of that domain
contains part of the conserved domain d (shared
by all Pol's)
downstream of the invariant Mg
2+-binding motif
NADFGDG. Its C-terminal half corresponds to conserved
domain e
(
38) and contains the eukaryotic consensus motif
P.I.KP..LW.GKQ
shared by all eukaryotic and archaeal Pol's (Fig.
2).
This two-hybrid
interaction is in excellent agreement with the
high-resolution
structure now available for Pol II (
8),
where the 516-to-639
fragment coincides almost exactly with a spatial
module (positions
508 to 663) called pore 1. As seen in Fig.
4, this is
the main
Rpb1p region contacted by Rpb8p, and the latter subunit
contacts
no other subunit of the core enzyme. However, Rpb8p also
contacts
Rpb1p at Lys738 and Asp974, two positions that are not
included
in the two-hybrid interacting domain but could play an
important
role in the overall folding of
Rpb1p.
The P.I.KP..LW.GKQ consensus motif noted above corresponds to a short
18-
19-
16 fold on the pore 1 module (Fig.
3 and
4)
and is
located across Rpb8p on the spatial structure. The invariant
glycine of
that motif marks the beginning of
16 and is mutated
in the
temperature-sensitive Pol I mutant
rpa190-G728D (or
rpa190-
3)
(
41). The effect of this
mutation is not alleviated by increasing
the gene dosage in
RPB8 but, as already mentioned, is strongly
suppressed by
RPB6 (Fig.
3). Taken together, these data strongly
suggest
that Rpb8p, Rpb6p, and the pore 1 module form a "ménage
à
trois" in terms of functional and structural
interactions.
Site-directed mutagenesis of Rpb8p: effect on growth and on
two-hybrid interactions.
Seventeen conserved positions scattered
on the spatial structure and on the amino acid sequence of Rpb8p were
submitted to site-directed mutagenesis, yielding 30 mutants that were
tested for growth by plasmid shuffling in strain YGVS043
(34). As summarized in Fig. 1, 12 of these mutagenized
positions are invariant (or highly conserved, L38) in all eukaryotes
analyzed so far (F10, L38, P48, Y95, M97, G99, Y115, S117, G119, G120,
L121, L122). The remaining five positions (F6, P17, D53, R124, N131)
were chosen because they are also highly conserved in eukaryotes but
not in the S. pombe sequence. Keeping in mind that the
S. pombe subunit does not support growth in S. cerevisiae (35, 40), these positions were
mutated to match the corresponding S. pombe amino acid.
Most of the
rpb8 mutations were phenotypically
indistinguishable from the wild-type allele, indicating a remarkable
tolerance
of Rpb8p to amino acid changes, even when strictly invariant
positions
are replaced in a nonconservative way (e.g.,
rpb8-P48V,
rpb8-Y95E,
rpb8-M97K, or
rpb8-Y115E). While this mutagenesis was by no means
exhaustive, the spatial distribution of lethal (
rpb8-S117K,
rpb8-S117D,
rpb8-G119D,
rpb8-G120K,
rpb8-G120D) or conditionally lethal (
rpb8-L38D,
rpb8-G99D,
rpb8-G99K,
rpb8-L122D) mutations is clearly nonrandom,
as shown
in Fig.
4. With one exception (
rpb8-L38D), these mutations
are spatially close to each other and are located opposite the
main
Rpb1p interface, close to the P.I.KP..LW.GKQ consensus formed
by
18,
19, and
16 on Rpb1p. On Rpb8p, they correspond to the
6 (G99)
and
8 (S117) strands and to a highly conserved region
immediately
after
8, defined by the
119GGLLM
123 signature
shared
by all Rpb8p.
6 forms a
-addition motif with the
18 of
Rpb1p
(
8). These critical positions therefore appear to
define the
main interface between Rpb8p and Rpb1p.
rpb8-L38D
is the only
critical mutation located away from Rpb1p. This mutation is
likely
to disrupt the
4-
5-sheet motif and could thus strongly
affect
the overall folding of
Rpb8p.
These mutants were also examined for their effect on two-hybrid
interactions between Rpb8p and the largest subunit of Pol's
I
(Rpa190p), II, (Rpb1p) and III (Rpc160p). As shown in Fig.
5,
there is
a good correlation between their growth pattern and their
two-hybrid
response. The nine point mutants with complete or conditional
lethality
were all strongly impaired in their interaction with
one
(
rpb8-S117K,
rpb8-G120D,
rpb8-L122D),
two (
rpb8-S117D) or
all three
(
rpb8-L38D,
rpb8-G99D,
rpb8-G99K,
rpb8-G119D,
rpb8-G120K)
largest
subunits. In contrast, the phenotypically silent mutations
rpb8-R124A,
rpb8-N131K, and
rpb8-Ä
21 (deleted
-loop)
(
40)
fully retained the two-hybrid interaction pattern of
the wild-type
allele.
Rpb8-L122D, corresponding to the
119GGLLM
123 invariant
motif, was investigated in more detail, in view of its striking Pol
III-specific defective response in the two-hybrid assay. This
specificity is reflected in vivo, as shown by the low steady-state
level in tRNA of
rpb8-L122D cells grown at 30°C or shifted
to
37°C (Fig.
6). Moreover, this mutant
has a high steady-state level
of 20S pre-rRNA, a property noted
previously for bona fide Pol
III mutants and probably reflecting a link
between Pol III and
rRNA maturation (
4). A close link
between growth and two-hybrid
interaction data are also suggested by
the properties of the human
and
S. pombe subunit in
S. cerevisiae, since the former complements
a
rpb8-
null mutant in vivo (except at high temperature) and
retains a
wild-type interaction with all three subunits (Fig.
2), while the
latter fails to complement at any temperature and
fails to interact
with Rpa190p and Rpc160p (
40).
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 6.
mRNA, tRNA, and rRNA levels in rpb8-L122D.
Steady-state levels of the 20S pre-rRNA, 18S mature rRNA,
tRNALeu3, and ACT1 mRNA analyzed by Northern
blotting. RNAs were extracted from exponentially growing cells of
YGVS043 (wild type) and YJFB025 (rpb8-L122D) shifted
from 25 to 37°C for 7 h in YPD liquid medium. The
oligonucleotide probes and Northern hybridization conditions were
previously described (4). The time that each sample was
taken (in hours) is shown at the top of the gel.
|
|
Ygr089p is a putative partner of Rpb8p.
As noted above, our
two-hybrid screen with pGBT9-RBP8 yielded two additional putative
partners of Rpb8p. Two independent clones encode Nup82p, a poorly
conserved but essential component of the yeast nucleopore
(18). They overlap between amino acids 457 and 566. Four
different clones encoding fragments of the orphan protein Ygr089p
overlapping between positions 497 and 685 were also selected (Fig.
7A). The fact that overlapping fragments
were selected in the screen implies that Rpb8p recognizes a specific protein domain on the target protein. However, this specificity is no
proof that the corresponding interactions are physiologically relevant.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 7.
Characteristics of the YGR089w gene
product. (A) Schematic representation of the four inserts recognized by
pGBT9-RPB8 in a two-hybrid test. The hatched area denotes a hydrophilic
C-terminal domain. The coordinates of the Ygr089p fragments encoded by
the clones selected in the Rpb8p two-hybrid screen are indicated. (B)
Cellular localization of the YGR089w gene product. Cell
fractionation was done as described in Materials and Methods. Extract,
crude cell extract; nucleus, P13 fraction; cytosol, S100 fraction.
Antibodies raised against Dolichol phosphate mannose synthase and
hexokinase I were used as markers of the endoplasmic reticulum-nucleus
and cytosolic fractions, respectively. Twenty micrograms of total
proteins was loaded in each case. (C) Growth pattern of the
ygr089w-::URA3 null mutants.
Cells of the isogenic strains YGVS043 (WT), YGVS045 (replacement of the
yeast Rpb8p by Hs8), YPR2 (ygr089w-), YJB021
(rpb8-G99K), YPR6 (ygr089w- and Hs8),
and YPR8 (ygr089w- rpb8-G99K) were
streaked on YPD and incubated for 3 days at 34°C.
|
|
YGR089w encodes a protein with a mass of 106 kDa and with no
obvious structural features except for its hydrophilic C end.
No
related gene product can be recognized from presently available
genomic
sequences, even for the ascomycetous yeast
S. pombe. A
Myc(13X)-tagged version of Ygr089p copurifies with the endoplasmic
reticulum-nucleus fraction and was excluded from the cytosol (Fig.
7B),
but attempts to localize the tagged protein more precisely
by
immunofluorescence have been inconclusive. The
ygr089w-
::URA3 null mutant is
phenotypically silent in terms of growth, respiration,
and competence
for meiosis (not shown). However, the fact that
it has a moderate but
distinct adverse effect on growth in an
rpb8-G99K context,
or when yeast Rpb8p is replaced by the human
subunit (Fig.
7C),
supports the idea that the Rpb8p-Ygr089p interaction
may be
physiologically
relevant.
| |
DISCUSSION |
In this study, we sought physiological partners of Rpb8p by
genetic screens based on extragenic suppression and on two-hybrid interactions. The putative partners thus identified include the largest
subunit of each nuclear Pol (Rpa190p, Rpb1p, and Rpc160p), the common
subunit Rpb6p, and more tentatively, the Nup82p nucleoporin and the
orphan gene product Ygr089p. The interaction with the largest subunits
is in excellent agreement with the high-resolution crystal structure of
yeast Pol II recently published by Cramer et al. (8). It
involves a spatial cluster of amino acids containing the invariant
119GGLLM123 motif, shared
by Rpb8p in all eukaryotes investigated so far. Mutants with a defect
in this interaction have a severe growth defect or are lethal.
Moreover, an rpb8-L122D mutant at the
119GGLLM123 motif
specifically fails to interact with Rpc160p and has a preferential Pol
III defect in vivo.
The target two-hybrid domain in the large subunits (domain e)
(38) contains an invariant P.I.KP..LW.GKQ motif, located
across the 119GGLLM123
Rpb8p signature, and essentially corresponds to the pore 1 module on
the Pol II crystal structure (8). Our data strongly
suggest that similar contacts holds Rpb8p to Rpa190p and Rpc160p on Pol
I and Pol III. This interaction must be evolutionarily robust, since
the yeast and human subunits, differing by 107 out of 150 amino acids,
are largely indistinguishable in their ability to interact with all
three RNA Pol's, as documented by heterospecific complementation and
two-hybrid data and by the intact subunit composition of the purified
chimeric Pol I. On the other hand, minor differences between the target
domain on Rpa190p, Rpb1p, and Rpc160p may account for the Pol
III-specific response noted above for rpb8-L122D and for
Pol-specific effects also observed when replacing Rpb8p by the S. pombe subunit (40).
A moderate increase in the gene dosage of RPB6 suffices to
suppress the temperature sensitivity defect associated with the human
Rpb8p-like subunit (Hs8). Moreover, this also suppresses rpa190-G728D, a Pol I mutant located at the invariant Glycyl
of the Rpb8p target motif P.I.KP..LW.GKQ. An in vivo connection between Rpb6p and Rpb8p is thus clearly established by these data. Since RPB6 acts in a dose-dependent way and not as a result of a
mutational change, the suppressible mutants are likely to affect the
assembly or stability of Pol's rather than their activity. Rpb6p and
Rpb8p do not contact each other on the spatial model of Pol II, where they are actually separated by a large spatial domain corresponding to
the pore 1 module (8). Thus, Rpb8p, Rpb6p, and the pore 1 module may form a functionally integrated domain on the Pol II
structure, with structural changes at the Rpb8p side
(rpa190-G728D or the replacement of Rpb8p by the human Hs8
subunit) ultimately altering the folding of Rpb6p and perhaps
facilitating its dissociation form the core enzyme. Alternatively, we
also note that the present crystal structure lacks the entire
N-terminal half of Rpb6p. The latter is highly disorganized both in the
Pol II crystal data (8, 9) and in the NMR structure of the
human subunit (11) and essentiallly contains acidic amino
acids that could form a flexible helix stretching far out of Pol II and
thus possibly contacting Rpb8p. This domain is essential for growth in
S. cerevisiae (29) and is as uniquely
eukaryotic as Rpb8p itself (23). It is phosphorylated at
the N-terminal Ser of the human subunit (19), but the S2A
mutant fully retains its ability to support growth in S. cerevisiae.
How do these data relate to the essential role played by Rpb8p in
eukaryotic transcription, keeping in mind that this subunit is shared
by all three Pol's and is uniquely eukaryotic? One clue may be given
by the fact that Rpb8p is targeted to the pore 1 module, initially
recognized as eukaryotic specific on the basis of sequence alignments
(domain e; 38) and having different foldings in the
bacterial and yeast enzyme (8). Cramer et al. (8, 9) have speculated that pore 1 could facilitate the entry of nucleoside triphosphates and allow the 3' end of the nascent transcript to move backward in halted elongation complexes. Interestingly, the
P.I.KP..LW.GKQ motif located opposite Rpb8p in the pore 1 fold (and
mutated in the Pol I rpa190-G728D mutant) is also present on
archaeal Pol's, although the latter has no recognizable Rpb8p subunit (10, 23). This motif is thus unlikely to be a
mere docking site for Rpb8p. Moreover, the
119GGLLM123 motif of Rpb8p appears to form a flexible or partly disorded small loop across the
P.I.KP..LW.GKQ motif. It would be worth examining in more detail
how the corresponding rpa190-728D
(P.I.KP..LW.GKQ) and rpb8-L122D (GGLLM) mutants affect
transcriptional activity in vitro.
On the other hand, Rpb8p may not be directly required for the activity
of eukaryotic Pol's but could rather determine the stability of their
heteromultimeric structure or their correct nuclear assembly. As noted
above, the suppression of rpa190-728D by an
increased RPB6 gene dosage points indeed to such a stability or assembly defect. In a more speculative vein, the interaction noted
between Rpb8p and the Nup82p nucleoporin (18) could also reflect a role of Rpb8p in the nuclear transport or assembly of Pol's
I, II, and III. Finally, we note that most of Rpb8p is exposed to the
solvent in the three-dimensional model of Pol II, thus forming an
external surface available for nonessential interactions with other
nuclear components, such as the one tentatively suggested here for the
orphan yeast gene product Ygr089p.
| |
ACKNOWLEDGMENTS |
We thank O. Gadal for very useful suggestions, M. Riva and C. Carles for a sample of purified Pol I and for Pol I antibodies, A. Voutsina for S. pombe plasmids, M. Nomura for the
rpa190-3 mutant, and P. Cramer, D. A
Bushnell, and R. Kornberg for communicating their Pol II coordinates
prior to publication. We heartily thank sharp-eyed and patient referees
for improving our manuscript.
J.-F.B. had a Fellowship from the Fondation de la Recherche
Médicale, and F.N. held a European Marie Curie Fellowship. This work was partly funded by a Training and Mobility Program of the European Union (grant FMRX-CT96-0064).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Biochimie and Génétique Moléculaire, CEA/Saclay,
F-91191 Gif-sur-Yvette, France. Phone: 33.1.69 08 35 86. Fax:
33.1.69 08 47 12. E-mail: thuriaux{at}matthieu.saclay.cea.fr.
Present address: Department of Experimental Biology, University of
Jaén, Paraje las Lagunillas, E-23071 Jaén, Spain.
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Molecular and Cellular Biology, September 2001, p. 6056-6065, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.6056-6065.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
