Molecular basis of RNA polymerase III transcription repression by Maf1.
ABSTRACT RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
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ABSTRACT: Enhanced indexation is a structured investment approach that combines passive and active financial management techniques. We propose an enhanced indexation model whose goal is to maximize the excess return that can be attained with high reliability, while ensuring that the relative market risk does not exceed a specified limit. We measure the relative risk with the coherent semideviation risk functional and model the asset returns as random variables. We consider that the probability distributions of the index fund and excess returns are imperfectly known and belong to a class of distributions characterized by an ellipsoidal distributional set. We provide a game theoretical formulation for the enhanced indexation problem in which we maximize the minimum excess return over all allowable probability distributions. The variance of the excess return is calculated with a computationally efficient method that avoids model specification issues. Finally, we show that the game theoretical model can be recast as a convex programming problem and discuss the results of numerical experiments.Decision Analysis 06/2012; 9(2):146-155. · 2.14 Impact Factor
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ABSTRACT: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, the method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This 'spike' chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences, including global and largely uniform changes.Genome Research 04/2014; · 13.85 Impact Factor
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ABSTRACT: Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.PLoS Genetics 12/2014; 10(12):e1004789. · 8.17 Impact Factor
Molecular Basis of RNA Polymerase III
Transcription Repression by Maf1
Alessandro Vannini,1,3Rieke Ringel,1,3Anselm G. Kusser,1,3Otto Berninghausen,1George A. Kassavetis,2
and Patrick Cramer1,*
1Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universita ¨t
Mu ¨nchen, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
2Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA
3These authors contributed equally to this work
RNA polymerase III (Pol III) transcribes short RNAs
required for cell growth. Under stress conditions,
the conserved protein Maf1 rapidly represses Pol III
transcription. We report the crystal structure of
Maf1 and cryo-electron microscopic structures of
Pol III, an active Pol III-DNA-RNA complex, and
a repressive Pol III-Maf1 complex. Binding of DNA
and RNA causes ordering of the Pol III-specific sub-
complex C82/34/31 that is required for transcription
initiation. Maf1 binds thePol IIIclamp andrearranges
C82/34/31 at the rim of the active center cleft. This
impairs recruitment of Pol III to a complex of
promoter DNA with the initiation factors Brf1 and
TBP and thus prevents closed complex formation.
Maf1 does however not impair binding of a DNA-
RNA scaffold and RNA synthesis. These results
explain how Maf1 specifically represses transcrip-
tion initiation from Pol III promoters and indicate
that Maf1 also prevents reinitiation by binding Pol
III during transcription elongation.
The eukaryotic genome is transcribed by the multisubunit
enzymes Pol I, II, and III, which catalyze DNA-dependent
RNA synthesis. Pol III transcribes genes encoding short,
untranslated RNAs, including transfer RNAs, 5S ribosomal
RNA (rRNA), the spliceosomal U6 small nuclear RNA (snRNA),
and the signal recognition particle 7SL RNA. Pol III genes are
essential and involved in fundamental processes such as ribo-
some and protein biogenesis, RNA processing, and protein
transport. Pol III transcription is coregulated with Pol I activity,
accounting together for up to 80% of nuclear gene transcription
in growing cells (Paule and White, 2000; Grummt, 2003; Willis
et al., 2004). Pol III activity is a critical determinant of cell
Pol III is the most complex of the nuclear RNA polymerases.
It has a total molecular weight of around 700 kDa and comprises
17 subunits (Schramm and Hernandez, 2002). Five of its
subunits, Rpb5, 6, 8, 10, and 12, are common to Pol I, II, and
III. Subunits AC40 and AC19 are common to Pol I and III and
The two largest Pol III subunits C160 and C128 are homologous
to Pol II subunits Rpb1 and Rpb2, respectively, and form the
active center of the enzyme. Subunits C17 and C25 form
a subcomplex with homology to the Pol II subcomplex Rpb4/7
(Ferri et al., 2000; Jasiak et al., 2006; Sadhale and Woychik,
1994), whereas subunit C11 shares homology with Pol II subunit
Rpb9. The Pol III-specific subunits C82, C53, C37, C34, and C31
form two subcomplexes. The C53/37 subcomplex shows weak
homology to the Pol II initiation factor TFIIF and is involved in
promoter opening, elongation, termination, and reinitiation
(Cramer et al., 2008; Carter and Drouin, 2009; Kassavetis et al.,
2010; Landrieux et al., 2006), whereas the C82/34/31 subcom-
plex is involved in promoter recognition and initiation. C34 inter-
1995; Wang and Roeder, 1997; Werner et al., 1993) and is
involved in open complex formation (Brun et al., 1997).
To date, structural information on Pol III is limited to a cryo-elec-
tron microscopic (cryo-EM) map that revealed the approximate
location of the two Pol III-specific subcomplexes (Ferna ´ndez-
Tornero et al., 2007), a homology model for the 10-subunit
core enzyme, and the crystal structure of C25/17 (Jasiak et al.,
Rapid repression of Pol III transcription ensures cell survival
during stress (Warner, 1999). Pol III repression is mediated by
Maf1, a protein that is conserved from yeast to human (Pluta
et al., 2001; Upadhya et al., 2002). Maf1 represses Pol III in
response to DNA damage, oxidative stress, growth to
stationary phase, treatment with rapamycin or chlorpromazine,
and blocking of the secretory pathway (Upadhya et al., 2002;
Willis et al., 2004). In growing yeast, Maf1 is phosphorylated
and localized in the cytoplasm. Stress conditions lead to
Maf1 dephosphorylation and nuclear import (Oficjalska-Pham
et al., 2006; Roberts et al., 2006), which is directed by two
nuclear localization signal (NLS) sequences (Lee et al., 2009;
Moir et al., 2006). In the nucleus, Maf1 binds Pol III to prevent
its interaction with TFIIIB and promoters (Desai et al., 2005;
Moir et al., 2006; Roberts et al., 2006). Maf1 also binds Brf1,
a subunit of TFIIIB that resembles the Pol II initiation factor
Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc. 59
TFIIB (Desai et al., 2005). Maf1-mediated repression is associ-
ated with reduced Brf1 and Pol III occupancy at Pol III genes
(Oficjalska-Pham et al., 2006; Roberts et al., 2006). Similar
results have been obtained with human cells, establishing
Maf1 as a conserved global repressor of Pol III transcription
(Reina et al., 2006).
Here, we report cryo-EM structures of Pol III in its free form
and in complex with a DNA-RNA scaffold, assign the locations
of Pol III subunits, present the Maf1 crystal structure, and
combine the resulting information with a cryo-EM structure of
a Pol III-Maf1 complex. Together with functional studies, these
results establish the mechanism for Pol III transcription repres-
sion by Maf1.
RESULTS AND DISCUSSION
Pol III EM Structure Reveals C82/34/31 Mobility
We established a protocol for large-scale purification of Pol III
Procedures). Pure Pol III samples comprised all 17 subunits
(Figure 1A), were monodisperse, and appeared homogeneous
in EM with negative stain (Figure 1B). We collected high-quality
cryo-EM data after vitrification under native conditions. A recon-
struction of Pol III from 20,480 single particles led to a map at
21 A˚resolution (Figure 1E; Figure S1 available online; Experi-
mental Procedures) that generally agrees with the previously
published map (Ferna ´ndez-Tornero et al., 2007).
Pol II X-ray
Active center cleft
Rpb8 footC160 foot
Pol II X-ray
Figure 1. Cryo-EM Structures of Pol III and Pol III-DNA-RNA Complex
(A) SDS-PAGE of pure yeast Pol III. The identity of the 17 subunits was confirmed by mass spectrometry.
(B) EM micrographs of Pol III in negative stain (left) and vitrified ice (right). Scale bars represent 10 nm.
(C) Views of the Pol III reconstruction (first row) with corresponding raw single-particle images (second row), low pass-filtered single-particle images (third row),
class averages (forth row), and reference-free averages (fifth row).
(D) DNA-RNA scaffold used in complex formation.
shown as a ribbon model. White dashed lines indicate additional densities between the lobe and Rpb9 (C11), attributed to the C53/37 subcomplex, and between
the clamp and Rpb5, attributed to the C82/34/31 subcomplex, that gets ordered in the DNA-RNA complex.
See also Figures S1 and S4 and Movie S1.
60 Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc.
The 12-subunit Pol II crystal structure (Armache et al., 2005)
was unambiguously fitted to the EM map (Figure 1E). After that,
two densities remained that could not be assigned to Pol III-
specific insertions or residues lacking from the Pol II structure,
one at the polymerase lobe and one on top of the clamp
(Figure 1E). Densities at the lobe and clamp were attributed to
subcomplexes C53/37 and C82/34/31, respectively (Ferna ´n-
dez-Tornero et al., 2007). The density at the lobe was fitted with
a homology model of the C53/37 dimerization module based on
the structure of the related A49/34.5 module in Pol I (Geiger
et al., 2010) (Figure 2). The location of C53/37 agrees with the
previously reported association of C53/37 with C11 (Che ´din
tional density at the clamp accounts only for part of the 138 kDa
subcomplex C82/34/31, indicating flexibility (Figure 1E).
Nucleic Acid Binding Restricts C82/34/31
To see how nucleic acid binding influences the Pol III structure,
we determined the cryo-EM structure of a Pol III complex with
a minimal DNA-RNA scaffold (Figure 1D; Experimental Proce-
dures). This complex mimics an active elongation complex
(Brueckner et al., 2007). A reconstruction at 19 A˚resolution
struction revealed density for nucleic acids in the cleft, but also
a structural ordering of the C82/34/31 subcomplex, giving rise
to an extended density between the top of the clamp, the
Rpb5 jaw, and C25/17 (Figures 1E and 2A; Figure S2).
A continuous density between the clamp and the jaw could be
fitted with the crystal structure of the human C82 homolog
(S. Fribourg, personal communication) (Figure 2). A prominent
density remained, forming a suspension over the cleft from the
clamp to the protrusion (Figures 1E and 2A–2C). This density
Outline view from
the C25/17 side
Figure 2. Subunit Architecture of Pol III
(Geiger et al., 2010), the human C82 homolog crystal structure (blue; S. Fribourg, personal communication), and the two C34 WH domain crystal structures
(purple) are shown as molecular surfaces. Fitted structures are shown low-pass filtered tothe same resolution than the EM map. The 12subunit Pol II X-ray struc-
ture (Armache et al., 2005) is shown as a green ribbon.
(B) Close-up views of Pol III-specific subunits fitted into the cryo-EM envelope of the Pol III-DNA-RNA complex. Terminal extensions of the C53/37 dimerization
module are highlighted in red.
(C) Location of Pol III-specific subunits on the Pol II structure. The view is related to the one in (A) by a 90?rotation around a horizontal axis.
(D) Location of subunits of the C82/34/31 subcomplex within Pol III.
(E) Domain organization of Pol III-specific subunits. Based on homology modeling (C53, C37), crystallography (C34), or HHPred and secondary structure predic-
See also Figures S2 and S4 and Movie S1.
Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc. 61
was assigned to subunit C34 since its two lobes fitted the struc-
tures of two winged helix (WH) domains in C34 (PDB codes 2dk5
and 2dk8), and since C34 crosslinks to promoter DNA around
position ?21 (Bartholomew et al., 1993), which is adjacent in
the homologous Pol II promoter complex model (Kostrewa
et al., 2009). The remaining globular density between the clamp
and C25/17 (Figure 2; Figure S2) was assigned to C31 since this
position explains the known interactions of C31 with subunits
C160, C82, C34, and C17 (Che ´din et al., 1998; Geiduschek
and Kassavetis, 2001; Schramm and Hernandez, 2002), the
requirement of the adjacent zinc site Zn8 in C160 for C82/34/
31 binding (Werner et al., 1992), and association of C31 with
Pol III after dissociation of the C82/34 heterodimer (Lorenzen
et al., 2007). Thus, all Pol III subunits were assigned to EM densi-
ties consistent with known subunit interactions.
Globular Structure of Maf1
To elucidate Pol III repression by Maf1, we determined the Maf1
structure by X-ray crystallography (Experimental Procedures).
Limited proteolysis of recombinant S. cerevisiae and human
Maf1 revealed two flexible regions, a mobile insertion and an
acidic C-terminal tail (Figure 3A). A human variant that lacked
both mobile regions crystallized. The structure was solved by
bromide phasing and refined to a free R factor of 21.2% at
1.55 A˚resolution (Table 1). Maf1 forms a globular structure
with a central five-stranded antiparallel b sheet that is flanked
by one helix on one side and three helices on the other
(Figure 3B). The Maf1 fold is frequently observed, but not in
proteins involved in transcription (Holm and Park, 2000; Krissinel
and Henrick, 2004). The structure shows that the previously
defined conserved sequence boxes A, B, and C (Desai et al.,
2005; Pluta et al., 2001; Reina et al., 2006) do not correspond
to structural modules or defined surface patches (Figure 3C).
Thus, functional data for Maf1 deletion variants must be re-eval-
uated in light of the structure. The Maf1 structure is conserved
among eukaryotes, since hydrophobic core residues are
conserved from yeast to human (Figure 3A).
Regulated Maf1 Localization
The Maf1 crystal structure reveals that the two NLS sequences
(yeast residues 205–208 and 328–332; Moir et al., 2006) are
surface accessible (Figure 3B). The C-terminal NLS (Ct-NLS) is
located between strands b4 and b5, and the N-terminal NLS
(Nt-NLS) is part of the directly adjacent mobile region
(Figure 3B).The adjacent location suggests thatphosphorylation
of the mobile insertion regulates nuclear localization by masking
the NLS sequences (Lee et al., 2009; Moir et al., 2006). This
mechanism is apparently conserved from yeast to human,
although the phosphorylation sites within the mobile insertion
differ (Dephoure et al., 2008; Lee et al., 2009; Moir et al., 2006;
Shor et al., 2010). The Ct-NLS and adjacent residues form the
only positively charged region on Maf1 (Figure 3F). Several point
mutants that lead to defects in phosphorylation, growth on glyc-
erol at 37?C, or Pol III repression (Moir et al., 2006; Roberts et al.,
2006) are exposed around the mobile insertion (Figure 3E, resi-
dues labeled in red and pink).
Maf1 Rearranges C82/34/31
To investigate how Maf1 binds yeast Pol III, we prepared full-
length recombinant yeast Maf1 and a variant that lacked both
with Pol III that could be purified by size-exclusion chromatog-
raphy (Figure 3D, lanes 3 and 4). Maf1 binding was specific, as
human Maf1 did not bind yeast Pol III (data not shown).
Thus, the two mobile regions are not required for Pol III binding,
and the human Maf1 crystal structure is relevant for under-
standing the Pol III-Maf1 interaction in the yeast system. We
collected cryo-EM data of the pure Pol III-Maf1 complex and
used 16,974 particles to obtain a reconstruction at 18.5 A˚resolu-
in the Pol III-DNA-RNA complex (Figure 4C; Figure S5).
Maf1 was assigned to a new density on top of the clamp with
the help of difference maps (Figure 4; Figure S3). The Maf1 X-ray
structure fitted this density well (Figures 4A and 4C; Figure S3).
To provide additional support for the Maf1 location, we labeled
the C-terminal hexahistidine tags on Maf1 and the Pol III subunit
C128 with Ni-NTA-Nanogold and located the labels by 2D cryo-
EM image analysis (Experimental Procedures). The locations of
the labels were consistent with Maf1 binding on top of the clamp
domain (Figure 4B). This location also agreed with published
biochemical and genetic interactions of Maf1 with the N-terminal
region of C160, which forms most of the clamp (Boguta et al.,
1997; Oficjalska-Pham et al., 2006; Reina et al., 2006)
(Figure 4D). Further consistent with this location, C160, C82,
and C34 are the key interacting partners of Maf1 in the yeast
interactome (Gavin et al., 2006).
Maf1 partially overlapped with the assigned locations of the
second WH domain in C34 and with C82 and C31 in the Pol III-
DNA-RNA complex (Figures 4C and 4E). Consistently, the C82/
34/31 density in the Pol III-Maf1 complex differed from that in
the Pol III-DNA-RNA complex. Most of the density assigned to
the C34 WH domains in the Pol III-DNA-RNA complex was
absent in the Pol III-Maf1 complex, indicating a Maf1-dependent
ties assigned to C31 and C82 apparently shifted toward the
Rpb5 jaw (Figures 4C and 4F; Figure S3). The differences in
the EM structures are visualized in a side-by-side comparison
and a movie (Figure S4; Movie S1).
Maf1 Impairs Closed Promoter Complex Formation
To analyze how the structural changes induced by Maf1 could
repress Pol III transcription, we modeled the Pol III-Brf1-TBP
closed promoter complex. Brf1 resembles the Pol II initiation
factor TFIIB in its N-terminal region but contains a specific
C-terminal extension that binds TBP (Figure S5) (Khoo et al.,
1994). We combined the Pol II-TFIIB-TBP closed promoter
complex model (Kostrewa et al., 2009) with the structure of
TBP bound to the Brf1 C-terminal residues 437–507 (Juo et al.,
2003). Comparison of the resulting model with the EM densities
revealed that C34 was well positioned for interacting with the
Brf1 N- and C-terminal regions (Figure 5A), consistent with
published data (Khoo et al., 1994, Andrau et al., 1999; Brun
et al., 1997; Kassavetis et al., 2003). In the Pol III-Maf1 complex,
62 Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc.
C34 adopts a different position that is apparently incompatible
with Brf1 interaction, suggesting that Maf1 impairs Pol III recruit-
ment to Brf1-containing promoters (Figures 5A and 5B).
To test this model, we investigated by size-exclusion chroma-
tography whether the Pol III-Maf1 complex can bind to
a preassembled functional Brf1-TBP-DNA promoter complex
mobile insertion (Nt-NLS)
mobile insertion (Nt-NLS)
mobile insertion (Nt-NLS)
mobile insertion (Nt-NLS)
Sc Maf1 fl
Sc X-tal Maf1
Figure 3. Maf1 Crystal Structure
(A) Amino acid sequence alignment of Maf1 from Homo sapiens (H.s.), Schizosaccharomyces pombe (S.p.), and Saccharomyces cerevisiae (S.c.). Secondary
structure elements are indicated (cylinders, a helices; arrows, b strands). Identical and conserved residues are highlighted in green and orange, respectively.
The mobile insertion (human residues 36–82, yeast residues 36–224) includes proteolytic cleavage sites (this work), phosphorylation sites (Dephoure et al.,
2008; Lee et al., 2009; Moir et al., 2006), and the N-terminal NLS (Nt-NLS). The C-terminal NLS (Ct-NLS) is indicated. Dashed lines indicate regions absent
from the crystal structure. The crystallized protein is a human Maf1 variant comprising residues 1–35 and 83–205.
(B) Two views of a ribbon model of the Maf1 crystal structure. Secondary structure elements are labeled according to (A).
(C) Maf1 ribbon model with the conserved boxes A, B, and C highlighted in blue, purple, and rose, respectively.
(D) Purification of Pol III-Maf1 complexes. Two hundred microrams of Pol III and a 5-fold molar excess of full-length yeast Maf1 or a variant comprising residues
1–35 and 225–345 (lane 1) were incubated for 20 min at 20?C, subjected to gel filtration, and analyzed by SDS-PAGE. Lanes 2, 3, and 4 show Pol III, the Pol III
complex with the Maf1 variant, and the Pol III complex with full-length Maf1, respectively.
(E) Surface conservation of Maf1. Identical and conserved residues are highlighted in green and yellow, respectively. Mutations at residues labeled in red, pink,
and wheat show severe, mild, or no phenotypes, respectively (Dephoure et al., 2008; Moir et al., 2006; Roberts et al., 2006).
(F) Surface charge distribution of Maf1. Red, blue, and white areas indicate negative, positive, and neutral charge, respectively.
Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc. 63
(Kassavetis etal.,2005).WeusedU6snRNApromoter DNAfrom
position ?40 to +20 relative to the transcription start site +1
(Figure 6A, closed scaffold). Whereas free Pol III stably bound
the Brf1-TBP-DNA complex, the Pol III-Maf1 complex did not,
even when a 5-fold molar excess was used (Figure 6B, lanes
3, 5). When we repeated the experiment with a mismatched
bubble region at positions –11 to +2 (Figure 6A, bubble scaffold),
the same result was obtained (Figure 6E, lanes 6, 7). Further,
preassembled Pol III-Brf1-TBP-DNA complex did not bind
Maf1, even when a 5-fold molar excess was used (Figure 6B,
lane 4). Thus, the interactions of Pol III with Maf1 and a Brf1-
TBP-DNA complex are mutually exclusive, showing that Maf1
impairs formation of a closed promoter complex. This is consis-
tentwith evidencethatMaf1preventsPolIIIpromoter interaction
(Desai et al., 2005; Moir et al., 2006; Roberts et al., 2006).
Maf1 Does Not Inhibit Pol III Activity
The above model predicts that Maf1 inhibits binding of promoter
DNA over the active center cleft, but not in the cleft. To test this,
we compared pure Pol III and Pol III-Maf1 complexes in an initi-
ation factor-independent transcription assay using a 30-tailed
DNA template and a priming RNA dinucleotide (Bardeleben
et al., 1994). Consistent with the model, both complexes were
equally active in RNA synthesis, and an excess of Maf1 or DNA
did not change activity (Figure 6C). We also performed RNA
extension assays using a minimal DNA-RNA scaffold (Damsma
and Cramer, 2009). The presence of Maf1 neither prevented
scaffold binding nor elongation to the end of the template, and
this was independent of the order of factor addition (Figure 6D).
To rule out that nucleic acids displace Maf1 from Pol III or
whether Pol III binds Maf1 and nucleic acids simultaneously. Pol
III-Maf1 complexes with tailed template or bubble scaffold could
be purified, independent of the order of addition (Figure 6E).
Thus, Maf1 prevents neither nucleic acid binding in the active
center nor RNA synthesis. The observation that Pol III can simul-
taneously bind Maf1 and nucleic acids suggests that the
increased Maf1 occupancy at Pol III genes under repressive
conditions (Geiduschek and Kassavetis, 2006; Oficjalska-
Pham et al., 2006; Roberts et al., 2006) is due to Maf1 binding
to elongation complexes. Pol III in such Maf1-containing elonga-
tion complexes would be unable to reinitiate, explaining the
observation that Maf1 represses multiple-round transcription
by Pol III (Cabart et al., 2008).
Our results converge with published data on the mechanism of
Pol III-specific transcription repression by Maf1. Cellular stress
leads to dephosphorylation of a mobile surface region in Maf1
that unmasks adjacent NLS sequences, leading to nuclear
import of Maf1. In the nucleus, Maf1 binds free Pol III at its clamp
domain and rearranges the C82/34/31 subcomplex. This impairs
Pol III binding to a TBP-Brf1-promoter complex and specifically
abolishes initiation from Pol III promoters, which require Brf1.
leaving activity intact but preventing reinitiation. Since Pol III
genes are short and elongation is fast, this leads to rapid shut-
down of all Pol III transcription.
Pol III Preparation
The Saccharomyces cerevisiae strain NZ16 (Lannutti et al., 1996), carrying the
gene for an N-terminally His6-FLAG4-RET1-tagged C128 subunit on theparent
plasmid pYE(CEN3)30 was grown to OD600= 6–7 at 30?C in YPD media in
a 200 L fermenter (Infors ABEC). Cells were lysed by bead beating in ice-
cooled buffer A [200 mM Tris-HCl (pH 8.0), 500 mM (NH4)2SO4, 10 mM
MgCl2, 10% glycerol, 10 mM b-mercaptoethanol, 1 mM PMSF, 1 mM benza-
midine, 200 mM pepstatin, 60 mM leupeptin]. Subsequent steps were per-
formed at 4?C. Glass beads were separated by filtration, and the lysate was
cleared by centrifugation (60 min, 8000 g, Sorvall SLA-1500). A whole-cell
extract was obtained after centrifugation at 125,000 g for 90 min (Beckman
Ti45) by separation of the clear upper-middle phase from the turbid lower
phase. The supernatant was processed by step-wise ammonium sulfate
precipitation. Thirty-five percent (NH4)2SO4was added, and the sample was
stirred for 30 min and cleared by centrifugation (60 min, 8000 g, Sorvall
SLA-1500). The supernatant was precipitated over night after addition of
70% (NH4)2SO4. The pellet was recovered by centrifugation (60 min, 8000 g,
Sorvall SLA-1500) and resuspended in 3 liters of buffer B (40 mM HEPES
[pH 7.8], 5 mM MgCl2, 10% glycerol, 1 mM EDTA, 10 mM b-mercaptoethanol,
Table 1. Maf1 X-Ray Diffraction and Refinement Statistics
Data SetNaBr SoakNative
Space group P 212121
Unit cell axis:
a, b, c (A˚)
7.7 (50.7)6.0 (39.2)7.0 (51.3)5.2 (58.9)
22.0 (2.5)22.7 (3.0) 22.0 (2.4)22.3 (1.2)
99.0 (99.5) 98.8 (99.4) 98.9 (99.5) 94.3 (87.4)
3.9 (4.0)3.8 (3.9)3.8 (4.0)3.0 (1.9)
Number of reflections 26,183
Number of atoms
B factors (A˚2)
Rmsd from ideal
Bond lengths (A˚)0.006
Bond angles (?)0.959
Rmerge= S jI ? < I > j/S j where I is the integrated intensity of a given
R = S kFobsj ? jFcalck/S j Fobsj. Rfreewas calculated with 5% of data
excluded from refinement.
aThe highest-resolution shell is shown in parentheses.
64 Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc.
Layer of cross-
section in (F)
Maf1 X-ray Protrusion
Maf1 X-ray (background)
(C128 side, cross-section)
Front view (close-up)
Density for the
C34 WH domains
in Pol III-Maf1
Figure 4. Cryo-EM Structure of the Pol III-Maf1 Complex
(A) Comparison ofcross-section of EM structures of thePol III-Maf1 complex(red) and thePolIII-DNA-RNA complex (blue) reveals an additional density for Maf1.
(B) Different views of reference projections of the Pol III-Maf1 3D reconstruction (top row), corresponding Nanogold-labeled particles used for alignment (second
row), raw Nanogold-labeled particles (third row), Nanogold locations (circles) on the Pol III-Maf1 structure (forth row), and surface representations of reconstruc-
tions with the C128 N terminus and the location of Maf1 indicated by white and yellow dots, respectively (bottom row).
(C) Fit of the Maf1 X-ray structure (red surface, low-pass filtered to the resolution of the EM map) to the Pol III-Maf1 EM map (red grid). For comparison, the cryo-
EM map of the Pol III-DNA-RNA complex is shown (blue).
(D) Ribbon representation of the Pol III-Maf1 complex. The PolII X-ray structure (Armache et al., 2005) is shown in green, and the Maf1 structurein red. The clamp
C160 residues 1–245 are yellow. The Pol III-Maf1 cryo-EM map is shown as a red mesh.
(E) Steric clash of Maf1 (red ribbon) with C34 (purple) and C82 (cyan) as observed in the Pol III-DNA-RNA complex.
(F) Comparison of cross-sections of EM structures of the Pol III-Maf1 complex (red) and the Pol III-DNA-RNA complex (blue) reveals a shift of the C82/34/31
subcomplex upon Maf1 binding.
(G) Close-up view of the region above the clamp. Parts of the C34 densities in the Pol III-DNA-RNA complex (blue) are absent in the Pol III-Maf1 complex (red).
See also Figures S3and S4 and Movie S1.
Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc. 65
1 mM PMSF, 1 mM benzamidine, 200 mM pepstatin, 60 mM leupeptin). The
sample was applied to a 250 ml Biorex resin column (Biorad). Bound proteins
were eluted with buffer C (buffer B + 500 mM KCl + 5 mM imidazole [pH 8.0]).
The eluting proteins were loaded onto a 12 ml Ni-NTA Agarose (QIAGEN)
column. Subsequent washing steps were performed with buffer C containing
10 mM imidazole and buffer D [40 mM HEPES (pH 7.8), 5 mM MgCl2, 250 mM
(NH4)2SO4, 10% glycerol, 10 mM imidazole, 10 mM b-mercaptoethanol, 1 mM
PMSF, 1 mM benzamidine, 200 mM pepstatin, 60 mM leupeptin]. Proteins
were eluted with buffer D containing 250 mM imidazole and loaded onto
a HiTrap Heparin 5 ml column (GE Healthcare) and fractionated by application
of a salt gradient from 250 to 1000 mM (NH4)2SO4with buffer E(40 mM HEPES
[pH 7.8], 5 mM MgCl2, 20% glycerol, 0.5 mM EDTA, 10 mM b-mercaptoetha-
nol, 1 mM PMSF, 1 mM benzamidine, 200 mM pepstatin, 60 mM leupeptin).
Pooled fractions eluting at 500 mM (NH4)2SO4were diluted 5-fold with buffer
E, loaded onto an anion exchange column (Mono Q 10/100 GL, GE Health-
care), and fractionated with a salt gradient from 50 to 1000 mM (NH4)2SO4in
buffer F (40 mM HEPES [pH 7.8], 1 mM MgCl2, 5 mM DTT). Pol III-containing
fractions eluted at 600 mM (NH4)2SO4, were pooled, diluted to a concentration
of 50 mM (NH4)2SO4, supplemented with a 10-fold molar excess of recombi-
nant full-length C53/37 heterodimer, and incubated for 60 min. The sample
was concentrated to 1 ml with an Amicon Ultra-4 centrifugal filter unit
(MWCO 10 kDA, Millipore) and applied to gel filtration chromatography on
a Superose 6 column (Superose 6 10/300 GL, GE Healthcare) with buffer G
[20 mM HEPES pH 7.8, 50 mM (NH4)2SO4, 100 mM MgCl2, 10 mM ZnCl2,
5 mM DTT]. Pol III-containing fractions were pooled, concentrated to
1mg/ml withan Amicon Ultra-4centrifugal filterunit (MWCO 10kDA, Millipore)
and flash frozen in liquid N2after addition of 10% glycerol.
Cryo-EM Structure Determinations
Purified Pol III was diluted to 0.1 mg/ml in buffer G and applied to glow-dis-
charged precoated carbon holey grids (Quantifoil R3/3, 2 nm carbon on top).
Samples were flash frozen in liquid ethane with a semiautomated controlled-
in liquid nitrogen until transfer to the microscope. Micrographs were recorded
under low dose conditions of ?15 e/A˚2on a FEI Tecnai Spirit microscope
operating at 120 kV, equipped with a LaB6filament and a Gatan side entry
Pol III-Brf1-TBP closed
promoter complex model
Pol II X-ray
Pol III-Brf1-TBP closed
promoter complex model
Pol II X-ray
Figure 5. Mechanism of Pol III Repression by Maf1
(A) Model of the Pol III-Brf1-TBP-DNA closed promoter complex. The Pol II structure is silver, the C34 WH domains are magenta, the Brf1 N-terminal domain is
green, the Brf1 C-terminal domain is orange, TBP is dark purple, and the closed promoter DNA is cyan/blue. The model is based on the homologous Pol II-TFIIB-
TBP-DNA closed promoter complex model (Kostrewa et al., 2009) and the Brf1-TBP-DNA structure (Juo et al., 2003).
(B) Schematic view of Maf1-dependent repression of the formation of a Pol III-Brf1-TBP-DNA closed promoter complex. Colors are as in (A).
See also Figure S5.
66 Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc.
cryoholder. Images were acquired at underfocus values in the range of 1.5–
4 mm on a 2k 3 2k FEI Eagle CCD camera applying a pre-exposure of
100 ms at a magnification of 90,0003, resulting in a pixel size of 3.31 A˚/px
on the object scale. Image-processing operations were carried out with
SPIDER (Frank et al., 1996). Initial particle selection was performed with
EMAN (Ludtke et al., 1999). Reference particles were picked manually to avoid
discrepancies due to defocus and ice differences. Automatically selected
particles were verified visually. Windowed particles were aligned to 83 projec-
tions of the Pol II X-ray structure (1Y1W, Gaussian low-pass filtered to 35 A˚),
which lacked the mobile OB and HRDC domains of Rpb4/7. Particle assign-
ment to the reference projections was evenly distributed, barring few overrep-
resented outliers that were limited to prevent predominant views (Figure S1).
Backprojection of the particle images with the angles from reference-based
alignment resulted in a reconstruction that showed additional densities at
the clamp and C25/17 and was used as a reference for 20 rounds of angular
refinement. Images were backprojected in real space with the refined angles.
The resulting reconstruction was Gaussian low-pass filtered to 25 A˚and used
as reference for another round of alignment and refinement, and this proce-
dure was iterated until convergence. A 21 A˚reconstruction of Pol III from
a data set of 20,480 particles was obtained. During early stage of refinement,
density for a complete C25/17 complex and other additional densities ap-
peared that could be confirmed by an independent 23 A˚reconstruction from
12,174 particles (data not shown). Projections of the 20,480 particle Pol III
reconstruction, as well as their corresponding particle averages, were
compared to averages resulting from a reference-free 2D alignment method
with the program refine2d (Ludtke et al., 1999). A high portion of similar aver-
ages showed that the alignment and refinement based on the reference struc-
ture was not significantly biased. A cryo-EM data set of Pol III, prepared as
above, incubated with a 3-fold molar excess of DNA-RNA scaffold, was
collected, and a reconstruction at 19 A˚resolution was obtained with 11,965
particles. A 18.5 A˚reconstruction of a size-exclusion purified Pol III-Maf1
complex (see preparation for interaction assays) was obtained from 16,974
particles. The resolution of the structures could not be improved when
96,944 particles collected on film at 200 keV with a FEI Polara microscope
Maf1 Crystal Structure Determination
DNA encoding S. cerevisiae or human Maf1 was PCR-amplified from genomic
DNA and cloned into pET-28b vector (Novagen) with the NdeI/NotI restriction
sites, resulting in a N-terminal hexahistidine tag. E. coli BL21 (DE3) RIL cells
Sc Maf1 fl
no Pol IIIno NTPs
+ 5x Maf1+ 10x Maf1
Pol IIIPol III-Maf1
+ 2x Scaffold+ 5x Scaffold + 10x Scaffold
Sc Maf1 fl
Pol III-Brf1-TBP Bubble sc. + Maf1
Pol III-Maf1 + Brf1-TBP Closed sc.
Pol III-Brf1-TBP Closed sc. + Maf1
Pol III-Brf1-TBP Closed scaffold
Pol III-Maf1 + Brf1-TBP Bubble sc.
Sc Maf1 fl
Pol III-Maf1 + Tailed tem.Pol III-Bubble sc. + Maf1Pol III-Maf1 + Bubble sc.Pol IIIBubble scaffold
Figure 6. Maf1 Impairs Closed Promoter Complex Formation but Not Pol III Activity
(A) Nucleic acid scaffolds.
(B) Competition assays reveal that Maf1 impairs binding of Pol III to a Brf1-TBP-DNA complex. Preassembled Pol III-Brf1-TBP-DNA or Pol III-Maf1 complexes
were incubated with a 5-fold molar excess of competing factor or complex as indicated and subjected to gel filtration, and the peak fraction was analyzed by
SDS-PAGE. In lanes 3, 4, and 6, the presence of DNA was revealed by the high A260/A280ratio (?1) compared to the A260/280ratio (?0.6) in lanes 2, 5, and 7.
(C) Factor-independent Pol III transcription assays. Preincubated Pol III-DNA (lanes 3–5) and Pol III-Maf1 complexes (lanes 6–8) efficiently transcribe the tailed
template (A). Addition of increasing amounts of Maf1 to preincubated Pol III-DNA complexes does not impair transcription (lanes 4 and 5). Increased amounts of
scaffold have no effect (lanes 6–8).
(D) RNA extension assay. The elongation scaffold (A) was efficiently transcribed to produce run-off product (+15) by Pol III upon addition of NTPs (lane 3).
Preincubation or addition of Maf1 (lanes 4 or 5, respectively) did not impair activity.
(E)PolIIIcansimultaneouslybindMaf1andnucleicacids.PreassembledPolIII-Maf1 andPolIII-DNA complexes wereincubatedwith5-fold molarexcessofDNA
or Maf1, respectively, and subjected to gel filtration, and the peak fraction was analyzed by SDS-PAGE and silver staining. Staining of a Pol III-Maf1 complex
(without DNA) is identical to that in lanes 4, 5, and 6.
Cell 143, 59–70, October 1, 2010 ª2010 Elsevier Inc. 67
(Stratagene) were transformed with the plasmid and grown in LB medium at
37?C to an OD600of 0.6. Expression was induced with 0.5 mM IPTG for
16 hr at 18?C. Cells were lysed by sonification in buffer H (50 mM HEPES
[pH 7.8], 0.5 M NaCl, 10 mM imidazole, 5 mM MgCl2, 10 mM EDTA, 10% glyc-
erol, 10 mM b-mercaptoethanol). After centrifugation, the supernatant was
loaded onto a 3 ml Ni-NTA column (QIAGEN) equilibrated with buffer H, but
20 mM imidazole. The column was washed with 20 column volumes (CVs)
and eluted with buffer H, but 300 mM imidazole. Proteins were purified by
anion exchange chromatography (Mono Q, GE Healthcare). The column was
equilibrated with buffer I (50 mM HEPES [pH 7.8], 5 mM MgCl2, 100 mM
EDTA, 10 mM b-mercaptoethanol, 10% glycerol), and proteins were eluted
with a linear gradient of 20 CVs from 10 mM to 1 M NaCl. After concentration,
the sample was applied to a Superdex-75 size-exclusion column (GE Health-
care) equilibrated with buffer L (25 mM HEPES [pH 7.0], 25 mM NaCl, 5 mM
DTT) for crystallization experiments or buffer M [50 mM HEPES (pH 7.8),
40 mM (NH4)2SO4, 100 mM MgCl2, 10 mM ZnCl2, 5 mM DTT] for binding exper-
iments.For partial proteolysis,100mlpurified Maf1at 1mg/ml weremixedwith
containing 1 mM CaCl2. Aliquots of 10 ml were taken at 1, 3, 5, 10, 30, and
60 min, and the reaction was stopped by addition of 5 3 SDS sample buffer
and incubation at 95?C for 5 min. Samples were analyzed by SDS-PAGE.
The N termini of digestion products were analyzed by Edman sequencing.
For crystallization, human Maf1 variant 1–35;83–205 was concentrated to
40 mg/ml. Crystals were grown within 2 days at 20?C in hanging drops over
a reservoir solution containing 50 mM MES (pH 6.0) and 175 mM sodium
oxalate. Native crystals were transferred into reservoir solution containing
25% glycerol and were flash cooled in liquid nitrogen. Crystals were soaked
for 0.5–2 min in a reservoir solution containing 25% glycerol and 0.5 M NaBr
and flash frozen in liquid nitrogen. Diffraction data were collected at 100 K
on a PILATUS 6M detector at the Swiss Light Source (SLS), Villigen,
Switzerland (Table 1). Three-wavelength anomalous diffraction data were
collected from abromide-soaked crystal. Data were processed with MOSFLM
(Leslie et al., 1986) and scaled with SCALA (Evans, 2007), and data quality was
assessed with Phenix.Xtriage (Adams et al., 2010). Program Phenix.HySS
(Adams et al., 2010) identified six bromide sites that were used for phasing
with program SOLVE (Terwilliger and Berendzen, 1999). Density modification
was carried out with RESOLVE (Terwilliger, 2003). The model was built with
COOT (Emsley and Cowtan, 2004) and refined with Phenix.Refine (Adams
et al., 2010) to a free R factor of 21% (Table 1).
M + 15 mM imidazole) with a 10-fold molar excess of recombinant full-length
Maf1. The complex was then incubated with a 20-fold molar excess of
to 1 ml with an Amicon Ultra-4 centrifugal filter unit (MWCO 10 kDA, Millipore)
and applied to gel-filtration chromatography on a Superose 6 column (Super-
ose 6 10/300 GL, GE Healthcare) with buffer G. Fractions were pooled and
samples prepared for cryo-EM as above. Cryo-EM data were collected as for
free Pol III but at an underfocus range of 3–4 mm to obtain high image contrast.
These were picked from the micrographs and aligned to projections of the Pol
III-Maf1 reconstruction. The strong signal of the Nanogold was dampened in
the images by manually applying a threshold to the histograms. The in-plane
rotation parameters resulting from the alignment were applied to the original
images, and the rotated images were compared to corresponding 2D surface
The length of the His6-tag and the ?0.9 nm linker between the gold cluster and
the nickel-NTA group of the Nanogold reagent give an expected mean vari-
ability of ?2 nm radius that was taken into account. The gold signal on the
N terminus of C128 displayed more apparent variability, which is explained
by the presence of four additional tandem FLAG sequences.
Interaction and Transcription Assays
vetis et al., 2005). Pol III-Brf1-TBP-DNA and Pol III-Maf1 complexes were
tively, in buffer M for 60 min at 4?C and purified by gel filtration (Superose 6 10/
300 GL, GE Healthcare) in buffer M. Purified complexes were then incubated
with a 5-fold molar excess of the competing factors, incubated in buffer M for
60 min at 4?C, applied again to gel filtration, and analyzed by SDS-PAGE. For
the nucleic acid binding assay, size exclusion-purified complexes were
analyzed by silver-stained gels. For factor-independent transcription assays,
1.5 pmol Pol III or Pol III-Maf1 complex were incubated for 30 min at 20?C
with 2 pmol or variable amounts of a pre-annealed tailed-template scaffold
(nontemplate DNA: 50-GGCTACTATAAATAAATGTTTTTTTCGCAACTATGTGT
TGATCAGCAGT-30; template DNA: 50-ACTGCTGATCATCTCTGTATTGTTTC
AAAAACATTTATTTATAGTAGCCTGCA-30) in the presence of 0.5 mM GpG
RNA primer. Complexes were incubated for 30 min at 20?C in the presence
of 0.3 mM ATP, GTP, CTP, NS [a-32P]UTP in 20 ml reaction mixtures containing
40 mM Tris-HCl (pH 8.0), 60 mM NaCl, 7 mM MgCl2, 7% glycerol, 5 mM DTT.
Reactions were stopped by addition of an equal volume of 23 loading buffer
(8Murea,23TBE) and incubationfor 5minat95?C.RNAproducts weresepa-
rated by denaturing gel electrophoresis and visualized with a Typhoon 9400
phosphoimager (GE Healthcare). For RNA extension assays, 5 pmol of Pol III
or Pol III preincubated (10 min at 20?C) with a 5-fold molar excess of Maf1
wasincubated for 30minat20?C with5pmol of apreannealedminimal nucleic
acid scaffold (template DNA: 30-TTACTGGTCCGGATTCATGAACTCGA-50;
nontemplate DNA: 50-TAAGTACTTGAG-30; RNA: 50-FAM-UGCAUUUCGAC
CAGGC-30). Maf1 was added at a 5-fold molar excess, followed by incubation
for 5 min at 20?C. For RNA elongation, complexes were incubated for 10 min
with 1 mM NTPs at 28?C in transcription buffer (60 mM ammonium sulfate,
20 mM HEPES [pH 7.6], 8 mM magnesium sulfate, 10 mM zinc chloride, 10%
glycerol, 10 mM DTT). Reactions were stopped and RNA products were
separated and visualized as above.
The coordinate file and structure factors for the Maf1 crystal structure were
deposited in the Protein Data BBank under accession code 3NR5. The EM
structures of Pol III, the Pol III-DNA-RNA complex, and the Pol III-Maf1
complex have been deposited in the EMDB database under accession codes
EMD-1753, EMD-1754, and EMD-1755, respectively.
Supplemental Information includes five figures and one movie and can be
found with this article online at doi:10.1016/j.cell.2010.09.002.
We thank R. Beckmann, T. Becker, C. Ungewickel, J. Bu ¨rger, and T. Mielke for
help withE.M. We thank A. Imhof (Zentrallabor fu ¨r Proteinanalytik) and T. Fro ¨h-
lich (Laboratory for Functional Genome Analysis) for mass spectrometry. We
acknowledge the crystallization facility at the department of E. Conti at the
Max Planck Institute of Biochemistry, Martinsried. We thank D. Deak for help
with figure preparation. A.V. was supported by a European Molecular Biology
Organization long-term fellowship and by the European Union training
program Marie Curie (MEIF-CT-2006-040653). P.C. was supported by the
Deutsche Forschungsgemeinschaft, the SFB646, the TR5, the Nanosystems
A.V. prepared Pol III complexes, A.V. and A.G.K. determined EM structures,
R.R. prepared and crystallized Maf1, R.R. and A.V. determined the Maf1
X-ray structure, R.R. and A.V. conducted functional assays, G.A.K. advised
on Pol III preparation, A.V., R.R., A.G.K., and P.C. wrote the manuscript, and
P.C. designed and supervised research.
Received: May 3, 2010
Revised: July 6, 2010
Accepted: August 11, 2010
Published: September 30, 2010
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