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Article https://doi.org/10.1038/s41467-023-37109-4
Structural basis of Ty1 integrase tethering
to RNA polymerase III for targeted
retrotransposon integration
Phong Quoc Nguyen
1,2,6
, Sonia Huecas
1,6
, Amna Asif-Laidin
3,6
,
Adrián Plaza-Pegueroles
1
,BeatriceCapuzzi
3
,NoéPalmic
3
, Christine Conesa
4
,
Joël Acker
4
, Juan Reguera
2,5
,PascaleLesage
3
&
Carlos Fernández-Tornero
1
The yeast Ty1 retrotransposon integrates upstream of genes transcribed by
RNA polymerase III (Pol III). Specificity of integration is mediated by an
interaction between the Ty1 integrase (IN1) and Pol III, currently unchar-
acterized at the atomic level. We report cryo-EM structures of Pol III in com-
plex with IN1, revealing a 16-residue segment at the IN1 C-terminus that
contacts Pol III subunits AC40 and AC19, an interaction that we validate by
in vivo mutational analysis. Binding to IN1 associates with allosteric changes in
Pol III that may affect its transcriptional activity. The C-terminal domain of
subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore,
providing evidence for a two-metal mechanism during RNA cleavage. Addi-
tionally, ordering next to C11 of an N-terminal portion from subunit C53 may
explain the connection between these subunits during termination and reini-
tiation. Deletion of the C53 N-terminal region leads to reduced chromatin
association of Pol III and IN1, and a major fall in Ty1 integration events. Our data
support a model in which IN1 binding induces a Pol III configuration that may
favor its retention on chromatin, thereby improving the likelihood of Ty1
integration.
Most living organisms harbor transposable elements (TE) that, upon
mobilizationwithin the genome, participate in the adaptative response
to environmental changes1. As mobile genetic elements, TEs also
represent a potential threat for genome integrity and,accordingly, can
cause human pathologies including cancer and aging2–4.Toreplicate
and yet minimize genetic damage to its host, TEs have evolved the
capacity of integrating into specific regions of the genome with mini-
mal effects on cell function, generally through tethering to different
cellular machineries that operate on the DNA5. Long-terminal repeat
(LTR) retrotransposons constitute a group of TEs that, like
retroviruses, replicate by reverse transcription of their mRNA in a
double-stranded DNA (cDNA) that is subsequently integrated into the
genome by their own integrase. Ty1 is the most active and abundant
LTR-retrotransposon in Saccharomyces cerevisiae6,7.Ty1preferentially
integrates into nucleosomes within the first kilobase upstream of
genes transcribed by RNA polymerase III (Pol III)8,9.
Pol III transcribes short genes encoding for untranslated RNAs
such as transfer RNAs (tRNA), the 5S ribosomal RNA (rRNA) and the
U6 spliceosomal RNA. Pol III is composed of 17 subunits that are
organized into four architectural units10–12. Within the main unit,
Received: 4 March 2022
Accepted: 2 March 2023
Check for updates
1
Centro deInvestigaciones Biológicas Margarita Salas, CSIC, 28040 Madrid,Spain.
2
Aix-Marseille Université, CNRS, AFMB UMR 7257, 13288Marseille, France.
3
Université Paris Cité, IRSL, Inserm, U944, CNRS, UMR7212, 75010 Paris, France.
4
UniversiteParis-Saclay, CEA, CNRS, Institute for Integrative Biology of the
Cell (I2BC), 91198 Gif-sur-Yvette, F rance.
5
INSERM, AFMB UMR7257, 13288 Marseille, France.
6
These authors contributed equally: Phong Quoc Nguyen, Sonia
Huecas, Amna Asif-Laidin. e-mail: pascale.lesage@inserm.fr;cftornero@cib.csic.es
Nature Communications | (2023) 14:1729 1
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termed core, Pol III-specific subunits C160 and C128 form the DNA-
binding cleft with the active site, while the assembly heterodimer
AC40/AC19 is shared with RNA polymerase I (Pol I), and peripheral
subunits Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12 are shared with Pol I and
RNA polymerase II (Pol II). The core is completed by subunit C11,
involved in transcriptional pausing and RNA cleavage, termination and
reinitiation13–15. Strong pausing induces enzyme backtracking asso-
ciated with fraying of a few nucleotides from the RNA 3′end, which, if
not cleaved by the C11 C-terminal domain13–15, may lead to enzyme
detachment from the DNA. Beside the core, the three remaining
architectural units are formed by other Pol III-specific subunits: (i) a
stalk including subunits C25 and C17, (ii) a nearby C82/C34/C31 het-
erotrimer sharing homology with transcription factor IIE (TFIIE), and
(iii) a TFIIF-like heterodimer formed by subunits C37 and C53, which
cooperate with C11 in termination and reinitiation14.Reinitiationisa
unique Pol III property whereby the terminating enzyme can restart
transcription on the same gene, assisted by initiation factors.
Genome-safe Ty1 integrationupstream of Pol III-transcribed genes
is mediated by an interaction between the Ty1 integrase (IN1) and the
Pol III AC40 subunit16,17. Contact with additional Pol III subunits
including C53, C34 and C31 has also been reported in vitro18. The IN1
N-terminal half retainsthe well-structured, phylogenetically-conserved
oligomerization and catalytic domains of retroviral integrases, while
the less-conserved C-terminal half is intrinsically disordered19.We
recently showed that a stretch ofresidues next to the C-terminal end of
IN1 is necessary and sufficient for association with AC40, IN1 recruit-
ment to Pol III-transcribed genes and Pol III-mediated integration16.
Nonetheless, the molecular details of this interaction have been
obscured by lack of structural studies.
In this work, we present cryo-EM structures of yeast Pol IIIbound
to IN1, in the absence and presence of a nucleic acid scaffold
mimicking the transcription bubble. We also perform structure-based
mutational analysis, both at residues involved in the interaction and at
allosteric Pol III regions that show an altered configuration in the
presence of IN1. Our results shed light on the mechanisms of Ty1
integration upstream of Pol III genes.
Results
Cryo-EM structures of Pol III bound to IN1
To gain structural insights into IN1 tethering on Pol III, we first
formed an in vitro complex between recombinant IN1 produced in
bacteria19 and the Pol III enzyme isolated directly from yeast20.The
two species interacted in a 1:1 stoichiometry as shown by native gel
electrophoresis (Supplementary Fig. 1a), demonstrating a direct
interaction between the two enzymes. This interaction can be sta-
bilized through mild crosslinking, despite the appearance of higher
order oligomers that do not interfere with subsequent cryo-EM
analysis. Using this sample, we reconstructed the cryo-EM structure
of the Pol III bound to IN1 at 2.6 Å resolution (Fig. 1a; Supplementary
Fig. 1b-d, map A; Supplementary Table 1). In parallel, we formed a
Pol III complex with a transcription bubble mimetic comprising an
11-nucleotide mismatched DNA region, where one of the mis-
matched strands is hybridized to a 10-nucleotide RNA molecule
(Fig. 1b, inset). Interestingly, IN1 is capable of interacting with this
Pol III-DNA complex as with free Pol III (Supplementary Fig. 2a),
suggesting that the interaction may occur at the periphery of this
multi-protein enzyme. This preparation was employed to obtain the
cryo-EM structure of the Pol III-DNA complex bound to IN1 at 3.1 Å
resolution (Fig. 1b; Supplementary Fig. 2b–d, map D; Supplemen-
tary Table 1). Additionally, we obtained the cryo-EM structure of the
Pol III-DNA complex in the absence of IN1 at 3.2 Å resolution (Fig. 1c;
Supplementary Fig. 3, map E; Supplementary Table 1), which we
used as a reference to assign map regions corresponding to IN1.
Despite overall similarities (Supplementary Fig. 4a, b), the cryo-EM
maps in the presence of IN1 exhibit remarkable differences
respect to those obtained in the absence of IN1 (Fig. 1;Supple-
mentary Fig. 4c), as described below.
Maps in the presence of IN1 reveal a piece of elongateddensity at a
creviceon the surfaceof subunit AC40, located between a hairpin-loop
(residues 108–130) and the C-terminal helix of this subunit (Figs. 1,2;
Supplementary Fig. 5a). In the absence of IN1, this crevice harbors a
molecule of detergent CHAPSO that we used to optimize cryo-EM grids
for samples containing nucleic acids (Supplementary Fig. 5a), as
reported21. We unambiguously assigned the elongated density from
maps in the presence of IN1 to an IN1 segment (residues 609–625)
proximal to its C-terminus (Fig. 2b). This segment contains the
tethering motif of IN1 involved in Ty1 integration targeting (residues
617–622), previously identified through mutational analysis and
referred to as targeting domain of Ty1 (TD1)16. Therefore, we hereafter
use TD1 to designate the whole AC40-binding segment. TD1 corre-
sponds to the sequence linking two nuclear localization signals (NLS1
and NLS2 in Fig. 2b, residues 596–598 and 628–630) that define a
bipartite NLS22,23. Our reconstructions lack density attributable to
either IN1 NLS, indicating that they are not conformationally con-
strained within the complex with Pol III and, therefore, may mediate
simultaneous binding to importin-αin vivo. M oreover, conservation of
TD1 in Ty2 and Ty4 retrotransposon integrases (Fig. 2b) suggests that
an equivalent mechanism may operate for their interaction with
AC4017 and preferential insertion upstream of Pol III-transcribed
genes7.
Our maps inthe presence of IN1showed no density for the rest of
IN1, including functional domains for Ty1 integration that locate at the
N-terminal half of the protein (Fig. 2b, NTD and CCD). This is likely due
to high flexibility of the IN1 linker region (residues 376–608), shown to
exhibit intrinsic disorder19, that connects functional domains with TD1.
Both in the presence and in the absence of IN1, our maps present an
elongated density nextto the AC40 hairpin-loop, also observed in a Pol
III pre-termination complex21. We tentatively attribute this density to
the N-terminus of the nearby subunit Rpb12 (Supplementary Fig. 5b).
Residual density also appears next to the Pol III stalk in all maps, likely
belonging to alternative conformations of a peripheral loop in subunit
C17 (Supplementary Fig. 5c).
Atomic details of the interaction between TD1 and AC40
In the structures of Pol III bound to IN1, TD1 adopts an extended
conformation that includes a helical turn along the AC40 crevice and
forms an intricate network of interactions with this subunit (Fig. 2b, c).
Residues W614, M619 and L622 in IN1 establish hydrophobic contacts
with P76, F315, I320 and the aliphatic chain of K316 in AC40. Addi-
tionally, R620 and S621 in IN1 establish one hydrogen bond (H-bond)
each with D111 and T114 in AC40, respectively. Importantly, K617 in IN1
appears as a central residue coordinating three salt bridges with D109,
D111 and E312 in AC40. The interaction between Pol III and TD1 is
reinforced by contacts with subunit AC19, where residues T136 and
K140 in this subunit establish an H-bond and a salt bridge with W614
and D612 in IN1, respectively. Our structural observations correlate
remarkably well with reported mutational analysis of IN1, showing that
individual mutations in residues K617, S621 or L622 disrupt the inter-
action with AC40 and alter Ty1 integration upstream of Pol III-
transcribed genes16. This study also showed that mutation of residues
N618 or E623, which in our structures point towards the solvent and
are not conserved in Ty4 integrase (Fig. 2b), have no effect on the
interaction between Pol III and IN1.
To investigate the role of individual AC40 residues in Ty1
integration, we produced single mutants at positions that appear
critical according to our structures. As AC40 is essential, we
checked that these mutations did not affect significantly cell growth
or AC40 protein levels (Supplementary Fig. 6a, b). We previously
showed that mutations altering the interaction between AC40 and
IN1 do not affect Ty1 integration frequency but induce a
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 2
Content courtesy of Springer Nature, terms of use apply. Rights reserved
redistribution of integration events into subtelomeric loci16. For all
mutants, we observed a less than twofold decrease in retro-
transposition frequency of a Ty1-his3AI reporter element expressed
on plasmid from the GAL1 promoter24 (Supplementary Fig. 6c),
similar to that observed for the AC40 Schizosaccharomyces pombe
ortholog (AC40sp), which behaves as a loss-of-interaction mutant17.
In a qualitative PCR assay to monitor in vivo Ty1 insertion events
(Fig. 2d), the SUF16 tRNA gene was previously identified as a hotspot
of Ty1 integration in the presence of wild-type AC40, while the HXT
subtelomeric genes were preferred target sites when the interaction
was compromised using AC 40sp or Ty1 elements mutated in TD116,17.
In comparison to the wild-type protein, AC40 mutants D111A, E312A
or K316E induced a significant reduction of Ty1 integration events
upstream of SUF16, associated with a sharp increase in Ty1 inte-
gration at HXT loci, similar to the AC40sp mutant. An equivalent
result was obtained when residue I320 in AC40 was mutated to
either tryptophan or glutamate, suggesting that a bulky or charged
residue at this position is sufficient to disrupt IN1 tethering to Pol III.
Notably, I320W was designed because AC40sp contains a trypto-
phan at this position. Although to a lesser extent than I320E, I320W
recapitulates the behavior of the AC40sp loss-of-function mutant.
Additionally, mutation of F315 into alanine has no impact on Ty1
integration, while the F315E mutant behaves as the AC40sp mutant,
highlighting the importance of a hydrophobic interaction between
this residue and W614 in TD1. In accordance, the W614A mutant in
IN1, lacking in our former IN1 mutational analysis16,impairedthe
two-hybrid interaction of TD1 with AC40 (Supplementary Fig. 7a, b).
This mutation also exhibited a reduction in integration events
upstream of SUF16 and an increase in Ty1 integration at HXT loci as
compared to wild-type IN1 (Fig. 2e), while it did not alter the overall
Ty1 integration frequency (Supplementary Fig. 7c), indicating that
this residue plays a major role in the interaction between Pol III and
IN1. An equivalent effect is observed for the M619A mutant, as well
as for previously-reported16 mutants K617A, S621A and L622A
(Fig. 2e). While alanine substitution of R620 in IN1 impaired the
interaction with AC4016, this mutation displayed an intermediate
phenotype with increased integration at subtelomeres, as well as at
SUF16 (Fig. 2e), suggesting a more complex role of R620, which is
not conserved in Ty4 integrase (Fig. 2b), in Ty1 integration target-
ing. We also prepared a double mutant of the AC19 subunit har-
boring the T136E and K140E changes, which do not affect cell
growth (Supplementary Fig. 8a). Our PCR-based integration assay
shows that the AC19 double mutant presents an intermediate inte-
gration profile that is similar to the wild-type upstream of SUF16 but
180º
180º
C37/C53 C11
C11
Rpb6
Rpb10
C160
C128
Rpb5
Rpb12
Rpb8
DNA-binding
cleft
C82/C34
C34
/C31
TFIIE-like
heterotrimer
TFIIF-like
heterodimer
Stalk
heterodimer
AC40/AC19
module
C25/C17
AC40/AC19
b
a
RNA
Template DNA
Non-template DNA
180º
180º
Top view Bottom view
+10 +15
Mg2+
+5–20 –15
Downstream DNAUpstream DNA
IN1
C11
C11
IN1
c
180º 180º
RNA
Template DNA
Non-template DNA
IN1
IN1
Pol III + IN1
Pol III-DNA + IN1
Pol III-DNA
C53
C53
RNA
Fig. 1 | Overall structures of Pol III in the presence and absence of IN1. Cryo-EM
maps (left and middle) and resulting structures (right) of Pol III bound to IN1 in the
absence(a) or in the presence (b) of a mismatched transcription bubble(inset), and
of Pol IIIcomplexed to themismatchedbubble in the absenceof IN1 (c). Allsubunits
and the mainstructural elements of PolIII are labeled.Mg2+ and Zn2+ ions are shown
as green and gray spheres in the right panels. Additionaldensities attributed to IN1
(blue) or Pol III (yellow and cyan for C11 and C53, respectively) are indicated, while
black ovals in panel ‘c’indicate missing densities in the absence of IN1. Filled
squares in the inset correspond to modeled nucleotides in the presence of IN1.
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved
increased integration at subtelomeres (Supplementary Fig. 8b).
This indicates some, but not essential, contribution of these AC19
residues to the interaction between Pol III and IN1.
We then tried to rescue the AC40sp loss-of-function mutant
phenotype. Superposition of the AC40sp structure25 onto our struc-
ture of Pol III bound to IN1 shows that all residues of AC40 involved in
TD1 binding are conserved with the exception of S. cerevisiae E312,
which in S. pombe corresponds to V322 (Supplementary Fig. 8c, d).
While conservative, changes from AC40sc residues F315 and I320 to
AC40sp residues I325 and V330 could also affect the interaction with
IN1. We produced the V322E mutation alone or combined with muta-
tions I325F and V330I in the AC40sp loss-of-interaction mutant but did
not observe recovery of Ty1 integration events at SUF16,withTy1
insertions still occurring preferentially at subtelomeric loci in both
mutants (Supplementary Fig. 8b). This indicates that differences
beyond the TD1-binding crevice, possibly involving Pol III rearrange-
ments, are relevant for Ty1 integration.
The C11 C-terminal Zn-ribbon inserts into the Pol III funnel pore
Besides a direct interaction between Pol III subunits AC40/AC19 and
the TD1 region of IN1, binding of the latter associates with allosteric
changes in the Pol III enzyme. A major change is observed in subunit
C11, involved in RNA cleavage13, as well as in termination and reinitia-
tion together with the C37/C53 heterodimer14. C11 comprises two Zn-
ribbons (residues 1–36 and 64–110) connected by a flexible linker
(Fig. 3a). In reported yeast Pol III structures12,26,whichalllackIN1,the
IN1-TD1
AC40
R611
K617
R620
L622
N618
E623
W614
M619
F315*
I320*
E312*
AC40 AC19
IN1-TD1
ba
AC40
AC19
Ct-helix
Hairpin-like
loop
IN1-TD1
AC40
Dimer Domain-2
1 76 220 295 335
4Fe4S-like
Ct-helixHairpin-like loop
Dim
c
IN1
NTD CCD CTD eCTD TD1
10 64 75 240 260 306 375 609 625
1635
NLS1 NLS2
IN1 SKKRSLEDNE-TEIKVSRDTWNTKNMRSLEPPRSKKRIHLIA (635)
IN2 SKKRSLEDNE-TEIEVSRDTWNNKNMRSLEPPRSKKRINLIA (655)
IN4 ---QNIEASGSPVQTVNKSAFLNKEFSSLNMKRKRKR····· (720)
::.:* . *.:.:: .*:: **: : :*:
595 600 605 610 615 620 625 630 635
W614*
K617*
L622*
S621*
K316*
M619*
Linker
D109
D111*
P76
D612
T136
K140
R620*
T114
V609
P625
d
e
WT W614A K617A M619A R620A S621A L622A
2.0
1.5
4.0
1.5
0.4
kb
ACT1
Ty1 SEO1
Ty1 SUF16
AC40 AC40sp D111A E312A K316E F315A F315E I320W I320E
1.5
1.0
3.0
1.0
0.4
kb
ACT1
Ty1 HTX
Ty1 SUF16
IN1
AC40
HIS3
HIS3
Fig. 2 | Interaction between AC40 and TD1. a Schematic representation of
AC40 structural domains and close-up view of the Pol III bound to IN1 structure
around AC40. Fully modeled TD1 is shown as a blue tube, while Pol III is shown as
surface. bSchematic representation of IN1-TD1 including local alignment with Ty2
and Ty4 integrases (IN2 and IN4), with close-up view of the model (blue) and
corresponding map (green mesh) around TD1. Hyphens and interpuncts in the
IN4 sequence correspond to gaps and insertions, respectively. NTD, CCD, CTD and
eCTD stand for N-terminal, catalytic core, C-terminal and extended C-terminal
domains,respectively. NLS1 and NLS2 indicate nuclearlocalizationsignals, noted in
bold fontin the IN1 sequence.cAtomic detailsof the interactionbetween AC40 and
IN1. H-bond, salt bridges and hydrophobic interactions are indicated with blue,
purple and green dotted lines, respectively. Asterisks denote residues mutated in
this study. dDetection of endogenous Ty1 insertions upstream of the SUF16 Pol III-
transcribed gene and the HXT subtelomeric genes (HXT13,HXT15,HXT16 and
HXT17) by PCRusing primers indicated with orange and greenarrows. Endogenous
Ty1 retrotransposition was induced in cells growing at 20 °Cduring 3 days in YPD
media. Total genomicDNA was extracted fromthree independentcultures. ACT1 is
genomicDNA quality control.eDetection of Ty1-HI S3de novo insertions generated
in cellstransformed bya plasmid expressing fromthe GAL1 promoter WT or mutant
Ty1-his3AI elements, bearing theindicated substitutions of conserved residues. Ty1-
HIS3 upstream of the SUF16 Pol III-transcribed gene and the SEO1 subtelomeric
gene were detected by PCR using a primer in HIS3 (red arrow) and a primer in the
locus of interest (green arrow).Ty1 retrotranspositionwas induced by growingcells
5 days at 20°C in the presence of galactose. Source data are provided as a Source
Data file.
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 4
Content courtesy of Springer Nature, terms of use apply. Rights reserved
C-terminal Zn-ribbon (C11-Ct) is either disordered or occupies a per-
ipheral location next to subunit Rpb5 (Supplementary Fig. 9a).
Accordingly, our yeast Pol III-DNA map, which lacks IN1, shows no
density for C11-Ct and half of the linker (Fig. 3a; Supplementary Fig.3a).
In contrast, our maps of Pol III bound to IN1 exhibit density for C11-Ct
within the Pol III funnel pore, a region that allows access to the active
siteoftheenzyme(Figs.1and 3a). An equivalent location for C11-Ct has
only been observed in recent structures of human Pol III in complex
with a transcription bubble27–29. Nevertheless, the human C11-Ct is
retracted away from the Pol III active site by about 9 Å, compared to
our structures of IN1-bound yeast Pol III (Supplementary Fig. 9b). Full
insertion of C11-Ct inside the funnel pore is expected when RNA clea-
vage is required following enzyme backtracking, as shown for
yeast TFIIS, the RNA cleavage factor in Pol II transcription30.This
unanticipated location of yeast C11-Ct in our IN1-containing structures
indicates that IN1 binding favors insertion of this domain within the
funnel pore.
Focused 3D classification of the Pol III bound to IN1 dataset
showed that approximately one third of the particles contain C11-Ct in
the funnel pore (Supplementary Fig. 1, map B), while the remaining
particles presented no density for this domain (Supplementary Fig. 1,
map C). Comparison of structures derived from these two maps
uncovered Pol III rearrangements for C11-Ct accommodation, mainly
involving opening of the C160 funnel and C128 fork domains using a
kink in the C160 bridge helix as hinge, associated with a fully ordered
C160 trigger loop (Fig. 3b).Thisloopisdisorderedinreportedapoor
elongating yeast Pol III structures12. Additionally, residues R698 and
Y701 in C128switch their conformation to allow access of the C11 acidic
loop, comprising residues 88-93 that are involved in RNA cleavage31,
into the active site (Fig. 3b, inset). Strikingly, focused classification of
the Pol III-DNA bound to IN1 dataset showed that virtually all particles
contain C11-Ct in the pore (Fig. 1b; Supplementary Fig. 2, map D). In
contrast, focused classification of the Pol III-DNA dataset, which lacks
IN1, produced the opposite result (Fig. 1c; Supplementary Fig. 3, map
E). In the structure of Pol III-DNA bound to IN1, the Pol III bridge helix
presents a straight conformation, associated to a conformational
change in the nearby C11 acidic loop, while the trigger loop is fully
ordered and retracted (Fig. 3c). In yeast Pol II, an equivalent config-
uration with a straight bridge helix and a locked trigger loop has been
observed in the reactivated intermediate of backtracking complexed
with TFIIS30 (Supplementary Fig. 9c, left panel).
The quality of our maps containing C11-Ct in the Pol III pore (maps
B and D) enabled precise building of all residues and metal ions in the
active site (Fig. 4a), where three catalytic aspartates (D511, D513, D515)
from subunit C160 coordinate a Mg2+ ion (MgA in Fig. 4b). In addition,
residue D91 in C11-Ct together with catalytic residues D511 and D513
coordinate a second Mg2+ ion (MgB in F ig. 4b)lying 3 Å away from MgA,
thus creating a composite active site with two metals as postulated for
Pol II30,32. Residue E92 in C11-Ct further coordinates MgB in the pre-
sence of nucleic acids, while its side chain is more flexible in their
absence (Fig. 4b). Consistently, residues D91 and E92 in C11 are
essential for RNA cleavage, while they are not involved in termination
or reinitiation13–15,33. In the presence of nucleic acids poor density
allows for fitting three base pairs of the RNA/DNA hybrid, in contrast
with our Pol III-DNA structure or that reported for the yeast Pol III
elongation complex12, where six and eight base pairs are respectively
observed for the hybrid (Fig. 4c). This indicates that, in the presence of
IN1, the interaction between Pol III and the hybrid is destabilized.
aZn-ribbon Linker
1 36 64 110
Zn-ribbon
C11
C11
cRNA/DNA hybrid
C160
Bridge helix
R
88
E92
C160
Trigger loop
Pol III + IN1 (from Map B)
Ordered C11-Ct
Pol III-DNA + IN1 (from Map D)
Ordered C11-Ct
versus
D91
Bridge helix
C160 Bridge helix (kinked)
Activ
v
v
e sit
es
es
e
C160 Trigger loop
C160 Funnel
loop & helices
C128
Fork loop
bPol III + IN1 (from Map B)
Ordered C11-Ct
Pol III + IN1 (from Map C)
Disordered C11-Ct
D91
E92
Y701
R698
versus
3’-RNA
C37
C37
C53
C53
C11-Ct
C11-Ct
(64-110)
C11-Linker
C11-Linker
(37-63)
C11-Nt
C11-Nt
(1-36)
C160-Jaw
C128-Lobe
C160C128
C128
Fork loop
C160
Funnel helices
Pol III-DNA + IN1 [colors] / Pol III + IN1
Pol III-DNA surface [Map E]
Fig. 3 | Structure of C11 in the presence of IN1. a Schematic representation of C11
and view around C11 in the structure of Pol III-DNA bound to IN1 (color ribbon)
fitted in the Pol III-DNA map lacking IN1 (graysurface). Subunits C11, C37 and C53
are shownin yellow, purple and blue, respectively. The structure of Pol III bound to
IN1 lacking DNA is shown in dark red, for comparison. bStructural comparison of
the Pol III bound to IN1 models derived from focused classification using a mask
around C11-Ct, showing presence (gray) and absence (green) of C11-Ct (yellow) in
the Pol III funnel pore. Arrows indicate differences between the structures. The
inset shows a close-up view around the C11acidic loop and thegating tyrosineY701
in subunit C128. cStructural superposition of Pol III bound to IN1 (gray) and Pol III-
DNA bound to IN1 (magenta) structures, with close-up views around the bridge
helix (upper panel) and C11-Ct (lower panel). Dotted circles mark a kink in the
bridge helix and remodeling of the C11 acidic loop, respectively.
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Superposition with the structure of bacterial RNA polymerase in the
pre-catalytic state34 allowed us to generate a model for RNA cleavage
by Pol III (Fig. 4d). Altogether, these observations suggest that, in
the presence of IN1, Pol III adopts a conformational state with C11-Ct
in the pore that might influence Pol III function in vivo. An RNA
extension assay shows a minor reduction of Pol III activity in the pre-
sence of IN1, especially for intermediate RNA products (Supplemen-
tary Fig.10a), which may reflect reduced Pol III dissociation from pause
sites. Moreover, a deletion mutant lacking C11-Ct (C11Δ71-110; C11ΔCt)
presents a twofold reduction in integration frequency and an altered
profile of integration at SUF16 relative to the wild-type (Supplementary
Fig. 10c-d). In contrast to AC40 or IN1 loss-of-interaction mutants, we
do not observe Ty1 integration at HXT subtelomeric loci, suggesting
that IN1 remains bound to Pol III in the C11ΔCt mutant. As this strain is
unable to grow at 20 °C (Supplementary Fig. 10b), which is optimal for
retrotransposition, our assays were performed at 25 °C, where growth
of this strain is partially affected and Ty1 overall retrotransposition still
occurs but at suboptimal levels. This could mitigate the effect of
C11ΔCt on Ty1 integration.
The C11 N-terminal Zn-ribbon and linker associate with C37/C53
In our structures, the C11 N-terminal Zn-ribbon (C11-Nt) is located
between the C160 jaw, the C128 lobe and subunit C37 (Figs. 3aand5a),
in an equivalent position to that observed in our Pol III-DNA structure
and reported structures in the absence of IN112.Wewereabletomodel
the entire C11 linker (residues 37-63) when C11-Ct is ordered (Supple-
mentary Fig. 1, map B; Supplementary Fig. 2, map D), independent of
the presence of nucleic acids. However, onlythe N-terminal thirdof the
C11 linker (residues 37–47) is visible when the C11-Ct is disordered
(Supplementary Fig. 1, map C; Supplementary Fig. 3, map E). In our
structures, this segment forms a β-strand extending a four-stranded β-
sheet in the C160 jaw domain (Fig. 5a). Only in the presence of IN1,
ordering of the entire C11 linker associates with the nearby appearance
of elongated densities that further extend the jaw β-sheet (Fig. 5a).
These densities correspond to a portion of the C53 N-terminal region
(C53-Nt, residues 1–274), which exhibits low-complexity11.Ourmapsin
the presence of IN1 allowed modeling of an extended loop of C53
followed by an α-helix (residues 195–227) and a short strand, which
become ordered next to the C11 linker. Ordering of C53 residues
195–227 in the same location has been observed in the Pol III pre-
termination complex21 (Supplementary Fig. 11a), while in other repor-
ted Pol III structures the entire C53-Nt is disordered12,26,35. However,
compared to the pre-termination complex, Pol III-DNA bound to IN1
exhibits an additional short strand and an open funnel that enables
access of C11-Ct into the pore (Supplementary Fig. 11a). Interestingly,
the C11-Nt and linker are essential for Pol III reinitiation15 and C11 acts
concertedly with the C37/C53 heterodimer during termination and
reinitiation14 and to prevent transcriptional arrest33 , suggesting that Pol
III might be more prone to terminate and/or reinitiate in the presence
of IN1. Moreover, a potential role in Ty1 integration has been raised for
the low-complexity region of subunit C5318.
To evaluate the biological relevance of this finding, we produced a
C53 deletion mutant lacking the entire low-complexity, N-terminal
region (C53Δ2-280; C53ΔNt), reported previously36. Chromatin
immunoprecipitation (ChIP) analysis shows that the C53-Nt truncation
leads to decreased occupancy of both Pol III and IN1 at two repre-
sentative Pol III-transcribed loci (Fig. 5b). Moreover, this mutant pre-
sents a growth defect that is more prominent at restrictive
temperatures (Supplementary Fig. 11b), as well as a 30-fold decrease in
integration frequency at 25°C as compared to the wild-type strain
(Fig. 5c). Such decrease is not observed in the presence of AC40sp loss-
of-interaction mutant (Fig. 5c), indicating that C53 plays a role in Ty1
integration that is different from that of AC40. Consistently, the
decrease in integration o f C53ΔNt affects Ty1 insertions both upstream
b
MgB MgA
D91
D511
D515
D513
MgA
D91
D511
D515
D513
Pol III + IN1 Pol III-DNA + IN1
3’-RNA
W
MgA
D91
D511
D515
D513
W
Backtracked
RNA [PDB -6RIN]
Pol III RNA cleavage (modelled)
C160
Active site
C11
Acidic loop
C160
Active site
C11
Acidic loop
Scissile bond
MgB
a
c
Pol III + IN1
[Map A - 2.6 Å]
Pol III-DNA + IN1
[Map D - 3.1 Å]
d
E92
D91 E92
D91 E92 E92
E92
MgB
Pol III elongation complex [PDB - 5FJ8]
RNA
C11-Ct
C11-Nt
C11-linker
Non-template DNA
Template DNA
Pol III-DNA
Pol III-DNA + IN1 [colors]
Fig. 4 | Pol III active site for RNA cleavage. a Active site model and map (blue
mesh) in the structures of Pol III bound to IN1 (left panel) and Pol III-DNA bound to
IN1 (rightpanel). bClose-up viewsof the active sitein the structuresof Pol III bound
to IN1 (left panel) and Pol III-DNA bound to IN1 (right panel). Green and red spheres
correspond to Mg2+ ions and water,respectively, while dotted lines indicate metal
coordination. cSuperposition between the structure of Pol III-DNA bound to IN1
(thick ribbon; RNA/DNA hybrid modelled from poor density, especially for RNA)
and those of Pol III-DNA ( green ribbon) and a canonical P ol III elongation complex
(grey ribbon; PDB 5FJ8). The C11 subunit in Pol III-DNA bound to IN1 is shown as
yellow surface, while the DNA template strand (TS) and non-template strand (NTS)
are blue and cyan, respectively, and the RNA is red. dModel of a pre-catalytic
complex for RNA cleavage using our Pol III bound to IN1 structure and an RNA
molecule from the superposed structure of bacterial RNA polymerase in pre-
catalytic state (PDB 6RIN).
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved
of Pol III-transcribed genes and at subtelomeric regions, in contrast to
what is observed in the presence of AC40sp (Supplementary Fig. 11c),
as reported18. Altogether, these results suggest that truncation of C53-
Nt leads to a defect in Pol III association to chromatin while free Pol III
remains bound to IN1, likely through subunits AC40/AC19, thus redu-
cing Ty1 integration. This is in line with pull-down assays showing that,
in this mutant, IN1 remains bound to Pol III despite lack of IN1 inter-
action with C3718.
Partial ordering of C34 and C31 regions in the DNA-binding cleft
Subunit C34 contains three winged-helix (WH) domains connected by
short linkers (Fig. 6a). The maps of Pol III bound to IN1 lacking nucleic
acids (maps A–C) exhibit weak globular density at the rim of the DNA-
binding cleft, between the C128 protrusion and the tip of the C160
clamp coiled-coil (Fig. 6a). The dimensions of this density and its
position next to the C34-WH3 domain led us to hypothesize that it
corresponds to the WH2 domain (residues 89–157) of this subunit.
Consistently, C34-WH2 occupies an equivalent position in the struc-
tures of Pol III initiation intermediates23–25 or Pol III complexed to
transcriptional repressor Maf137 (Fig. 6a). Focused 3D classification
confirmed the presence of this density in about 15% of the particles,
also showing an additional density next to C34-WH2 that might
account for the WH1 domain of this subunit (Supplementary Fig. 12a).
Superposition of our structures with that of Pol III in the presence of
TFIIIB26,35,38 shows that IN1 binding to Pol III is compatible with the
formation of the pre-initiation complex (Supplementary Fig. 12b). As
TFIIIB covers up to 60 base pairs upstream of Pol III-transcribed
genes39, thisis consistentwith the absence of Ty1 integration within the
first 80 base pairs upstream of tRNA genes8,9. These results could
indicate that, in the presence of IN1, a fraction of the Pol III enzyme
adopts a configuration that may favor interaction with TFIIIB, which
would facilitate promoter recruitment or retention.
Besides, our maps of Pol III bound to IN1 lacking nucleic acids
exhibit two globular pieces of density within the DNA-binding cleft.
One locates next to the C160 bridge helix, while the other lies near the
C128 fork and hybrid-binding domains (Fig. 6b). These densities, which
are apparent but unassigned in reported structures of free yeast Pol
III12,26, occupy the paths of downstream DNA and the RNA/DNA hybrid
(Supplementary Fig. 12c). While the shape of these densities hampers
model building, superposition with the s tructure of free human Pol III29
suggests that they may correspond to conserved regions of the C31
C-terminal tail (Fig. 6c). A third piece of elongated density occupying a
depression in the RNA exit channel, and extending into the hybrid-
binding region in the presence of IN1, could also correspond to a
segment of C31 as shown for human Pol III29 (Supplementary Fig. 12d).
Mutational analysis of the C31 C-terminal tail in yeast was either lethal
or produced a strong growth defect, reflecting a role in preventing
nucleic acid binding in free Pol III29. An equivalent role has been
assigned to the DNA-mimicking loop of Pol I40, which occupies the cleft
in the hibernating state of this enzyme41,42.
Discussion
Targeted DNA integration is crucial to maintain genome integrity. The
Ty1 retrotransposon takes advantage of the Pol III enzyme to integrate
Fig. 5 | Role of C53-Nt in Ty1 integration. a Schematic representation of C53 and
view of the Pol III-DNA bound to IN1 map around C53-Nt with th e resulting structure.
bQuantitative chromatin immunoprecipitation (ChIP) analysis of Pol III or IN1
enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from WT or
mutant rpc53Δ2-280 cells expressing a myc-tagged copy of C160 using anti-myc or
anti-IN1 antibodies is expressed as a valuerelativetothatoftheinput.Theunique
SCR1 gene or the 16 genes of the tDNA-Ile family are representatives of Pol III-
transcribed genes. The 18 S rDNA is transcribed by Pol I. GAL1 ORFservesasa
control. Values are mean ± SD, n= 3 experiments. p-values for C160-myc ChIP: SCR1,
0.0262; tDNA-Ile, 0.0025; 18S, 0.3379;GAL1, >0.9999. p-values for IN1 ChIP: SCR1,
0.0161; tDNA-Ile,0.0119;18 S,0.7700;GAL1,0.6495.cRetrotransposition frequency
of a chromosomal Ty1-his3AI reporter in WT and mutant rpc53Δ2-280 strains,
induced at 25 °C. Values are mean ± SD, n= 3 experiments, each performed with four
independent colonies. pvalue for C53Δ2-280 vs. C53: 0.0032. pvalue for AC40spvs.
C53: 0.4899. For all panels, p-values are *p<0.05;**p< 0.01; n s not significant. Two-
sided Welch’st-test was used. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
upstream of Pol III-transcribed genes, located in genome regions
devoid of essential genes, thus protecting the yeast genome. The
structures reported here provide evidence of primary IN1 binding
through its TD1 on Pol III subunits AC40 and AC19 (Figs. 1and 2). Our
structural and mutational analyses allowed definition of the precise
TD1 boundaries, comprising residues 609 to 625, and support that the
TD1-AC40 interaction is sufficient for IN1 tethering on Pol III and for
targeted integration (Fig. 2a, b).
Our maps show no density for the remaining of IN1, containing
functional domains for integration of the Ty1 cDNA. This supports a
scenario where the ~230 residue linker connecting the N-terminal half
to the C-terminus of IN1 (Fig. 2b), which is disordered19,providessig-
nificant flexibility between the Pol III-tethering and the DNA-integrating
modules of IN1. Ty1 integrates at DNA that is wrapped around the first
to the third nucleosomes located upstream of Pol III-transcribed
genes8,9,17 and, thus, a flexible linker is advantageous to reach varying
distances while keeping strong tethering on genome-attached Pol III.
This is in contrast with integrases from other LTR-retrotransposons or
retroviruses, all lacking a large disordered linker connected to their
respective targeting domains5. Retroviruses use transcriptional reg-
ulators of Pol II43, while the Ty3 integrase interacts with DNA-bound
TFIIIB in a configuration that blocks Pol III binding, thus hampering
transcription of the corresponding Pol III-transcribed gene44.
We show that IN1 binding associates with reordering of different
Pol III regions that are distant from the IN1 binding site. Since IN1
binding to AC40 slightly alters the conformation of this subunit, this
change is likely transmitted to other regions of Pol III (Supplementary
Fig. 4c), thus producing an allosteric effect. Unexpectedly, in the
presence of IN1, C11-Ct locates within the Pol III funnel pore, with its
acidic loop complementing and remodeling the Pol III active site
(Fig. 3). Notably, our structures show how a two-metal catalytic
mechanism could operate for RNA cleavage, thus providing a model
for this essential RNA polymerase activity (Fig. 4b, c). While this con-
figuration may interfere with the transcription process, it may as well
reduce Pol III pausing and its eventual detachment from DNA, as
suggested by RNA extension assays (Supplementary Fig. 10a). This
correlates with a mild reduction of integration frequency of a C11-Ct
deletion mutant (Supplementary Fig. 10c, d). In addition, a portion of
the low-complexity N-terminal region of subunit C53 orders next to the
C11 linker (Fig. 5a), similar to a Pol III pre-termination complex21
(Supplementary Fig. 11a). Our mutational and ChIP analyses show that
C53-Nt plays a role in Pol III binding to chromatin, which affects Ty1
integration while preserving the interaction between IN1 and subunit
AC40. Altogether, we speculate that IN1 binding induces a Pol III
configuration that increases its overall residence on the chromatin
without significantly affecting the overall RNA production by this
enzyme45. This is expected to enhance chances for IN1 to establish
productive integration complexes at nucleosomes upstream of Pol III-
transcribed genes and may, thereby, have contributed to the suc-
cessful propagation of Ty1 in the S. cerevisiae genome.
Finally, lack of density for the two NLSs flanking TD1 could allow
simultaneous binding of importin-αand Pol III by IN116.Moreover,
structural superposition with available structures shows no clash
between IN1 and TFIIIB. These observations are consistent with the
interaction between IN1 and Pol III taking place either in the nucleo-
plasm or while Pol III is bound on the DNA. Therefore, the TD1 motif
not only provides strong binding but also increased likelihood for the
tethering interaction to occur. This is especially relevant for the design
of new gene therapy vectors able to target safe regions of the genome.
The structures reported here represent a valuable tool in this respect,
as they allowatomic-level understanding of the molecular mechanisms
underlying targeted DNA integration upstream of genes transcribed by
Pol III.
Methods
Growth media, yeast strains, and plasmids construction
S. cerevisiae strains used in this study were grown using standard
methods and are listed in Supplementary Table 2. Plasmids were
aWH1 WH2
12 78 90 151 167 270
WH3C34
C34
1317
C34-WH2
C34-WH2
C34-WH3
C34-WH3
b
C128
Protrusion
C160
Clamp
Pol III + IN1 [Map A]
Pol III PIC [6EU0]
Pol III + IN1 [Map A]
Pol III-Maf1 [6TUT]
c
Bridge
helix
Globular
densities
Bridge
helix
K358
K360
K1391
R887
R888
R1373
K707
K1034
R472
R1028
RPC7-Ct (human)
C160
C128
Fork
Hybrid
binding
Fork
Hybrid
binding
Pol III + IN1 [Map A] Pol III + IN1 [Map A]
Free human Pol III [7D59]
Fig. 6 | Partial ordering of C34 and C31 in the presence of IN1. a Schematic
representation of C34 and viewof the Pol III bound to IN1 map around C34, where
the structure of Pol III from the reported Pol III PIC (PDB 6EU0; left) or Pol III-Maf1
complex(PDB 6TUT; right) has beenfitted. View of the DNA-binding cleft in the Pol
III bound to IN1 map with its corresponding model (b) or the structure of free
human Pol III (PDB 7D59; c).
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved
constructed using standard molecular biology procedures. Mutations
were introduced in plasmids with Q5® Site-directed mutagenesis
(NEB). All the constructs were validated by DNA sequencing (Eurofins
Genomics). All plasmids and primers used in this study are reported in
Supplementary Tables 3 and 4, respectively.
Construction of mutant strains
To construct RPC40 mutants, we used a S. cerevisiae yeast strain
(LV1690) deleted for its RPC40 genomic copy (AC40sc) and trans-
formed with a centromeric plasmid bearing Schizosaccharomyces
pombe RPC40 gene (pTET-HA-AC40sp, URA3) to ensure cell viability17.
Mutations were introduced in RPC40 carried on a centromeric plasmid
(pTET-HA-AC40sc, TRP1). Yeast strain was transformed with mutant
pTET-HA-AC40sc plasmids and cells were plated on DO-TRP for three
days at 30°C. Isolated colonies were selected and spread on DO+ 5-
FOA media (5-fluoroorotic acid) to counter-select yeast cells with
pTET-HA-AC40sp plasmid. Finally, scRPC40 mutant strains were vali-
dated by colony PCR, sequencing and western blots.
To construct V322E and V322E, I325F, V330I spRPC40 mutants,
the FYBL1-23D strain was transformed with pTET-HA-AC40spplasmids
harboring the mutations and cells were plated on DO-TRP for three
days at 30 °C. Then, the genomic RPC40 gene was deleted by gene
replacement using an rpc40::HphMX deletion cassette. Finally,
spRPC40 mutant strains were validated by colony PCR and sequencing.
To construct T136E, K140E RPC19 mutants, the LV33 strain was
transformed with pTET-HA-AC19 WT or mutant centromeric plasmids
and cells were plated on DO-TRP for three days at 30 °C. Then, the
genomic RPC19 gene was deleted by gene replacement using an
rpc19::NatMX deletion cassette and the RPC19 WT and mutant alleles
were validated by colony PCR and sequencing.
To construct RPC53 and rpc53Δ2-280 (C53ΔNt) strains, the
JC3787 strain harboring a chromosomal Ty1-his3AI reporter element
was transformed by pCW4 or pCW4mut centromeric plasmid
expressing RPC53 and rpc53Δ2-280, respectively36, and selected on DO-
LEU. The RPC53 endogenous copy was then deleted using a
rpc53Δ::KanMX deletion cassette. The strains were checked by colony
PCR and sequencing.
To construct the rpc11(1-70) (C11-ΔCt) mutant strain, the
LV1454 strain was transformed by a centromeric plasmid harboring
RPC11 (pAMA171) or rpc11Δ1-70 (pAMA173) and the RPC11 endogenous
copy was subsequently deleted in the transformants by gene repla-
cement using a rpc11Δ::KanMX deletion cassette. The strains were
checkedbycolonyPCRandsequencing.
Protein expression and purification
IN1 harboring a his tidine tag for purification followed by a sso7d tag for
solubility was produced as described19.Escherichia coli Rossetta(DE3)
cells transformed with the IN1-expressing plasmid were grown in TB
with ampicillin and chloramphenicol and grown at 25 °C to an OD600
equal to 0.6. Protein expression was induced by addition of 50 μM
IPTG and the cellswere grown overnight at 25 °C. Cells were harvested
at 1100g for 15min at room temperature, washed with PBS 1× and
weighed. Purification of IN1 was performed following described
procedures19.
For Pol III, S. cerevisiae strain SC1613, encoding a tandem affinity-
purification (TAP) tag at the C-terminus of subunit AC40, was provided
by Cellzome AG (Heidelberg, Germany). Pol III was purified as
described20 with some modifications. About 0.8 kg of cells were sus-
pended in buffer A (250mM Tris-HCl pH 7.4, 20% glycerol, 250 mM
(NH
4
)
2
SO
4
, 1 mM EDTA, 10mM MgCl
2
,10µMZnCl
2
,10mMβ-mer-
captoethanol) and lysed at 4 °C with glass beads in a BeadBeater
(BioSpec). After centrifugation, the supernatant was incubated over-
night at 4 °C with 6ml of IgG Sepharose (GE Healtcare), then the resin
was washed with 20 column volumes of buffer B (50 mM Tris-HCl pH
7.4, 5% glycerol, 250 mM NaCl, 5mM MgCl
2
,10µMZnCl
2
, 5 mM DTT)
and incubated overnight with tobacco etch virus (TEV) protease to
elute the protein by removal of partof the TAP-tag. The TEV eluate was
further purified using a Mono-Q (GE Healthcare) with a gradient from
buffer B to the same buffer containing 1 M NaCl. Pol III eluted at
~360 mM NaCl. The protein was concentrated to 7-8 mg/ml, frozen
with liquid nitrogen and stored at −80 °C until use.
Preparation of Pol III complexes with IN1
For Pol III bound to IN1, Pol III and IN1 both at 0. 5µM were incubated in
20 mM HEPES-NaOH, pH 7.5, 170 mM NaCl, 2 mM DTT for 30 min at
4 °C. The complex was crosslinked following described procedures
with minor modifications42.Briefly, the reaction was started by addi-
tion of 0.06%(v/v) of glutaraldehyde and, after 5 min incubation on ice,
the remains of the cross-linking agent were quenched by adding
50 mM glycine and incubating 5 min on i ce.
To prepare the mismatched transcription bubble, non-template
DNA (5′-GCAGCCTAGTTGATCTCATAGCCCATTCCTACTCAGGAGAAG
GAGCAGAGCG-3′), template DNA (5′-CGCTCTGCTCCTTCTCCTTT
CCTCTCGATGGCTATGAGATCAACTAGGCTGC-3′)andRNA(5′-AU
CGAGAGGA-3′) from Microsynth were incubated following described
procedures46. The Pol III-DNA complex was assembled by incubating
the transcription scaffold at equimolar amounts with the enzyme, at a
final concentration of 0.4 μM, for 1 h at 20 °C in buffer E (10 mM Hepes
pH 7.5, 150 mM NaCl, 5 mM MgCl
2
, 5 mM DTT). To obtain the Pol III-
DNA complex bound to IN1, the sample was then mixed with 0.6 μM
IN1 (final concentration) and incubated for 30 min at 20 °C in buffer E.
Cryo-EM grid preparation and data acquisition
The crosslinked complex between Pol III and IN1 was concentrated to
1µM, then 4 µl were applied to glow-discharged copper 300 mesh
C-flat 1.2/1.3 holey carbon grids (Protochips) in the chamber of a FEI
Vitrobot at 10°C and 100% humidity. The grids were blotted for 3.5s
with blotting force −5andvitrified by plunging into liquid ethane
cooled with liquid nitrogen. Movies were acquired on a FEI Titan Krios
(ThermoFisher) electron microscope at 300 keV using a K3 summit
(Gatan) directelectron detector operated in ‘super-resolution’mode at
defocus values between –1.0 and –2.8 µm and a physical pixel size of
1.06 Å, using EPU software. A total of 3321 non-tilted movies and 8900
movies tilted by 20° with 40 frames each were collected with an
accumulated total dose of 42.45 e-/Å2.
For Pol III-DNA inthe presence or in the absence of IN1, 3 µlofthe
sample at 0.4 or 0.1 µM were supplemented with 8 mM CHAPSO (final
concentration), applied to glow-discharged copper 300 mesh Quan-
tifoil R1.2/1.3 grids coated with continuous carbon, and incubated in
the chamber of a FEI Vitrobot at 24°C and 100% humidity for 1 min.
The grids were blotted for 4 sec with blotting force –5andvitrified by
plunging into liquid ethane cooled down to liquid nitrogen tempera-
ture. For Pol III-DNA bound to IN1, data were collected on a FEI Titan
Krios electron microscope operated at 300 kV, using a K3 summit
(Gatan) direct electron detector. Images were acquired at defocus
values between −1.0 and −2.8 μm and a pixel size of 1.085 Å. A total of
9327 movies with 40 frames each were collected with an accumulated
total dose of 45.12 e-/Å2. For Pol III-DNA in the absence of IN1, data were
collected on a Talos Arctica electron microscope operated at 200 kV,
using a Falcon III (FEI) direct electron detector in electron counting
mode. Images were acquired at defocus values varying between −1.2
and −2.6 μm at a pixel size of 0.855Å. A total of 2901 movies with 60
frames each were collected with an accumulated total dose of
30.96 e-/Å2.
Cryo-EM data processing
For the Pol III bound to IN1, movies were aligned and dose-corrected
using MotionCor247 as implemented in Relion 3.048. Global contrast
transfer function (CTF) parameters were estimated using GCTF49.
Around 1000 particles were picked manually to generate reference-
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved
free 2D classes that were used for template-based autopicking after
low-pass filtering to 20 Å, followed by estimation of local defocus of
the individual particles using GCTF. The remaining processing was
performed in Relion 3.1 (Supplementary Fig. 1). Threefold binned
particles were subjected to 3D classification and the best class with
885,560 particles was selected and subjected to another round of 3D
classification. The five best classes with 643,858 particles in total were
extracted with a 300 pixel box without binning and subsequently
refined, followed by two rounds of CTF refinement50 and Bayesian
polishing. Final post-processing was performed using automatic
masking and B-factor sharpening, resulting in a map at 2.5 Å resolution.
Five independent runs of 3D classification were performed using
masks for the AC40 subunit, the stalk, the protrusion plus clamp head,
C11-Nt and C11-Ct. For each case, the best classes were selected and
refined. The AC40 mask yielded a map at 2.6Å resolution with clear
density for TD1, while the C11-Ct mask produced maps at 2.9 and 2.8Å
resolution where this domain is present and absent, respectively.
For Pol III-DNA bound to IN1, movies were aligned and dose-
corrected using MotionCor2 as implemented in Relion 3.1, and their
CTF parameters were estimated using CTFFIND451. Approximately 550
particles were picked manually and reference-free 2D classes were
generated, five of which were used for template-based autopicking
after low-pass filtering to 20 Å. Approximately 2,437,000 particles were
automatically selected and extracted with a 300 pixel box using Relion
3.1, also employed for subsequent processing (Supplementary Fig. 2).
Three rounds of reference-free 2D classification yielded a stack of
469,620 good-quality particles that were refined using as reference the
EMD-3180 map filtered to 60 Å. The resulting map was used as a
reference for 3D classification to generate four classes. Two of these
classes showing Pol III-like shape were joined together (305,253 parti-
cles total) and refined to a resolution of 3.8 Å. The resolution of the map
was improved to 3.07 Å using particle polishing and CTF refinement.
For Pol III-DNA in the absence of IN1, movies were aligned and
dose-corrected using patch motion correction and their CTF para-
meters were estimated using patch CTF estimation, both as imple-
mented in cryoSPARC52. 667,000 particles were picked using blob
picker with a minimum and maximum diameter of 160 and 210Å,
respectively. After inspection, 527,000 particles remained and were
reference-free 2D classified. The final subset of 101,000 particles was
refined using homogeneous 3D refinement and our Pol III-DNA bound
to IN1 map, low-pass filtered to 60 Å, as starting model. Additional CTF
and non-uniform refinements yielded a map with a resolution of 3.2 Å.
Model building and refinement
The available structure of the Poll III bound to a transcription bubble
(PDB code: 5FJ8) was fitted in the 3D maps using UCSF Chimera53 and
employed as starting point for model building for Pol III subunits and
the nucleic acid scaffold. A homology model of the C11 C-terminal Zn-
ribbon was generated with Phyre254 and used as reference for model
building, while the C11 linker was manually modelled in Coot55. Avail-
able structures of Pol III complexed to melted DNA (PDB code: 6EU1)
and the Pol III-Maf1 complex (PDB code: 6TUT) were used as reference
to improve coordinates of the stalk C17/C25 heterodimer, the C82/
C34/C31 heterotrimer and the C37/C53 heterodimer. The nucleic acid
scaffold DNA model was adapted from a stalled Pol I elongation
complex (PDB code: 6H67). Segments corresponding to IN1 and C53-
Nt were manually-built de novo based only on our maps. The struc-
tures were refined using real-space refinement as implemented in
Phenix56.Refinement statistics are summarized in Supplementary
Table 1. Figures were prepared using PyMOL (Schrödinger Inc.) and
UCSF Chimera53.
RNA extension assay
The nucleic acid scaffold was prepared by mixing equal amounts of
template DNA: 5′-CGTAGCGGTATCGTGGTCGAGCGTGTCCTGGTCTA
G-3′, non-template DNA: 5′-CGCTCGACCACGATACCGCTACG-3′and
RNA: 5′-Alexa488-CGACCAGGAC-3′in 20 mM Tris, pH 7.5, 150 mM KCl,
heated to 95 °C and slow-cooled to 4°C. For RNA elongation, 100nM
Pol III was pre-incubated, when required, with 200 nM IN1 in 20 mM
Tris pH 7.5, 150 mM KCl, 5 µMZnCl
2
,10mMDTT for 10min atroom
temperature. Then 100nM of the scaffold was added and incubated
for another 10 min. To initiate the reaction, 1 mM of NTPs mix (Invi-
trogen) and 10 mM MgCl
2
was added and incubated at 37 °C. The
reaction was stopped by adding an equal amount of 2x RNA loading
dye (8 M urea, 2× TBE, 0.02% bromophenol blue, 10% (v/v) glycerol)
and heating to 95 °C for 5 min. The samples were loaded onto dena-
turing 20% polyacrylamide gel containing 7 M urea and visualized with
aFujifilm FLA-3000. For quantification, the percentage of each band
respect to the total signal per lane was analyzed with the ImageJ-NIH
software.
Ty1-his3AI transposition assays
To estimate the frequency of retrotransposition of pGAL1-Ty1-his3AI,
four independent transformants of each strain were grown to satura-
tion for 2 days at 30 °C in liquid SC-URA containing 2% raffinose. Each
culture was diluted thousand-fold in liquid SC-URA containing 2%
galactose and grown for 5 days to saturation at 20 °C, which is the
optimal temperature for Ty1 retrotransposition. Aliquots of cultures
were plated on YPD (100 μlat10
−5) and SC-HIS (100 μlat10
−2). Plates
were incubated for 3 days at 30 °C and colonies counted to determine
the fraction of [HIS+]prototroph.
Similar experiments were performed in C53 and C11 mutant
strains harboring a chromosomal Ty1-his3AI reporter, with the fol-
lowing differences. Four independent clones were grown at 30 °C to
saturation in liquid YPD. Each culture was diluted in YPD, a thousand-
fold for wild-type and C53 mutant strains and 250-fold for C11 mutant
strains, and grown to saturation at 25 °C. Aliquots of cultures were
plated on YPD (1 × 100 µl of a 1:20,000 dilution) and SC-HIS (2 × 2 ml).
Plates were incubated for 4–5 days at 30 °Cand colonies were counted
to determine the fraction of [HIS+] prototrophs.
A retrotransposition frequency was calculated as the median of
the ratios of number of [HIS+] cells to viable cells for each of the four
independent clones. Retrotransposition frequencies were defined as
the mean of at least three medians.
PCR assays for detection of Ty1 integration events
To detect endogenous Ty1 insertions, three colonies of each yeast
strain were inoculated overnight in YPD at 30 °C. The next day, each
culture was diluted thousand-fold in YDP (or 250-fold for C11
mutants) to induce Ty1 retrotransposition for 3 days at 20 °C (or for
5 days at 25 °C for the experiments with C53 and C11 mutants) and
total genomic DNA was extracted according to classical procedures57.
For the detection of Ty1-HIS3 insertions, pGAL1-Ty1-his3AI retro-
transposition was induced as described in the previous section and
total genomic DNA was extracted from yeast cultures grown at 20 °C
for 5 days. Double-strand DNA (dsDNA) concentration was deter-
mined using Qubit™fluorometric quantification (Thermo Fisher
Scientific). Ty1-HIS3 integrations upstream of tG(GCC)C (SUF16) were
amplified with PCR primers O-AL27 and O-AB91 and at the SEO1
subtelomeric gene with PCR primers O-AL27 and O-AL10. Endogen-
ous Ty1 integrations upstream of tG(GCC)C (SUF16) were amplified
with PCR primers O-AB46 and O-AB91 and at the HXT subtelomeric
genes (HXT13,HXT15,HXT16 and HXT17) with PCR primers O-AB46
and O-ABA27.
PCR reaction consisted of 30 ng of dsDNA (or 75 ng for detection
of insertions at HXT or SEO1), 5 μl Buffer 5×, 0.5 μldNTP10mM,
0.625 μl of each primer at 20 μM, 0,25 μl of Phusion DNA Polymerase
(Thermo Scientific) in a 25 μlfinal volume. Amplification was per-
formed with the following cycling conditions in ProFlex™PCR System
(Life Technologies) cycler: 98 °C 2 min, 30× [98 °C 10 s, 60 °C 30 s,
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
72 °C 1min], 72°C 5 min, and hold 4 °C. PCR products were separated
on a 1.5% agarose gel.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed as previously
described16 on crosslinked log-phase RPC53 or rpc53Δ2-280 cells
expressing a myc-tagged copy of C160, the largest subunit of Pol III.
After chromatin purification and solubilization, DNA-protein com-
plexes were immunoprecipitated using 25 µl of magnetic beads (Pan
Mouse IgG or Protein A Dynabeads, Invitrogen) coated with anti-myc
(1 µg, lab-made in mice from clone 9E10) or anti-IN1 antibodies (5 µlof
lab-made serum produced in rabbits immunized with recombinant IN
protein). The purified DNA samples were analyzed by quantitative real-
time PCR using the SYBR Green PCR master Mix kit (Thermo Fisher)
and an ABI PRISM 7500 (Applied Biosystems). The results were nor-
malized with the input DNA PCR signalsand indicated by relative IP in
the graphs. Values are the average of three independent experiments.
Statistical analysis
The two-sided Welch’sttest was used allowing unequal variance.
*p< 0.05; **p< 0.01; ***p< 0.001; ns, not significant. Statistical tests
were performed using GraphPad Prism version 9.0.0.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
The data that support this study are available from the corresponding
authors upon request. Electron density maps and associated coordi-
nates have been deposited in the Electron Microscopy and Protein
Data Banks, respectively, as follows: Pol III + IN1 from focused classifi-
cation on AC40 (EMD-14421/PDB 7Z0H), on C11-Ct with thisdomain in
the funnel pore (EMD-14469/PDB 7Z30), and on C11-Ct with this
domain disordered (EMD-14470/PDB 7Z31); Pol III-DNA + IN1 (EMD-
14468/7Z2Z); and Pol III-DNA (EMD-16299/PDB 8BWS). Source data are
provided with this paper.
References
1. Cosby, R. L., Chang, N.-C. & Feschotte, C. Host-transposon inter-
actions: conflict, cooperation, and cooption. Genes Dev. 33,
1098–1116 (2019).
2. Burns, K. H. Transposable elements in cancer. Nat. Rev. Cancer 17,
415–424 (2017).
3. Cardelli, M. The epigenetic alterations of endogenous retro-
elements in aging. Mech. Ageing Dev. 174,30–46 (2018).
4. Hancks, D. C. & Kazazian, H. H. Roles for retrotransposon insertions
in human disease. Mob. DNA 7, 9 (2016).
5. Sultana, T., Zamborlini, A., Cristofari, G. & Lesage, P. Integration site
selection by retroviruses and transposable elements in eukaryotes.
Nat. Rev. Genet. 18,292–308 (2017).
6. Carr, M., Bensasson, D. & Bergman, C. M. Evolutionary genomics of
transposable elements in Saccharomyces cerevisiae.PLoS ONE 7,
e50978 (2012).
7. Kim,J.M.,Vanguri,S.,Boeke,J.D.,Gabriel,A.&Voytas,D.F.Trans-
posable elements and genome organization: a comprehensive sur-
vey of retrotransposons revealed by the complete Saccharomyces
cerevisiae genome sequence. Genome Res. 8, 464–478 (1998).
8. Baller, J. A., Gao, J., Stamenova, R., Curcio, M. J. & Voytas, D. F. A
nucleosomal surface defines an integration hotspot for the Sac-
charomyces cerevisiae Ty1 retrotransposon. Genome Res. 22,
704–713 (2012).
9. Mularoni, L. et al. Retrotransposon Ty1 integration targets specifi-
cally positioned asymmetric nucleosomal DNA segments in tRNA
hotspots. Genome Res. 22, 693–703 (2012).
10. Fernández-Tornero, C. et al. Insights into transcription initiation and
termination from the electron microscopy structure of yeast RNA
polymerase III. Mol. Cell 25,813–823 (2007).
11. Fernández-Tornero, C. et al. Conformational flexibility of RNA
polymerase III during transcriptional elongation. EMBO J. 29,
3762–3772 (2010).
12. Hoffmann, N. A. et al. Molecular structures of unbound and tran-
scribing RNA polymerase III. Nature 528,231
–236 (2015).
13. Chédin,S.,Riva,M.,Schultz,P.,Sentenac,A.&Carles,C.TheRNA
cleavage activity of RNA polymerase III is mediated by an essential
TFIIS-like subunit and is important for transcription termination.
Genes Dev. 12,3857–3871 (1998).
14. Landrieux,E.etal.AsubcomplexofRNApolymeraseIIIsubunits
involved in transcription termination and reinitiation. EMBO J. 25,
118–128 (2006).
15. Mishra, S., Hasan, S. H., Sakhawala, R. M., Chaudhry, S. & Maraia, R.
J. Mechanism of RNA polymerase III termination-associated reini-
tiation-recycling conferred by the essential function of the N
terminal-and-linker domain of the C11 subunit. Nat. Commun. 12,
5900 (2021).
16. Asif-Laidin, A. et al. A small targeting domain in Ty1 integrase is
sufficient to direct retrotransposon integration upstream of tRNA
genes. EMBO J. 39, e104337 (2020).
17. Bridier-Nahmias, A. et al. Retrotransposons. An RNA polymerase III
subunit determines sites of retrotransposon integration. Science
348,585–588 (2015).
18. Cheung, S. et al. Ty1 integrase interacts with RNA polymerase III-
specific subcomplexes to promote insertion of Ty1 elements
upstream of polymerase (Pol) III-transcribed genes. J. Biol. Chem.
291,6396–6411 (2016).
19. Nguyen, P. Q. et al. Ty1 integrase is composed of an active
N-terminal domain and a large disordered C-terminal module dis-
pensable for its activity in vitro. J. Biol. Chem. 297, 101093 (2021).
20. Moreno-Morcillo, M. et al. Solving the RNA polymerase I structural
puzzle. Acta Crystallogr. D. Biol. Crystallogr. 70,2570–2582 (2014).
21. Girbig, M. et al. Architecture of the yeast Pol III pre-termination
complex and pausing mechanism on poly(dT) termination signals.
Cell Rep. 40, 111316 (2022).
22. Kenna,M.A.,Brachmann,C.B.,Devine,S.E.&Boeke,J.D.Invading
the yeast nucleus: a nuclear localization signal at the C terminus of
Ty1 integrase is required for transposition in vivo. Mol. Cell Biol. 18,
1115–1124 (1998).
23. Moore,S.P.,Rinckel,L.A.&Garfinkel, D. J. A Ty1 integrase nuclear
localization signal required for retrotransposition. Mol. Cell Biol. 18,
1105–1114 (1998).
24. Curcio, M. J. & Garfinkel, D. J. Single-step selection for Ty1 element
retrotransposition. Proc. Natl Acad. Sci. USA 88,936–940 (1991).
25. Heiss,F.B.,Daiß,J.L.,Becker,P.&Engel,C.Conservedstrategiesof
RNA polymerase I hibernation and activation. Nat. Commun. 12,
758 (2021).
26. Abascal-Palacios, G., Ramsay, E. P., Beuron, F., Morris, E. & Vannini,
A. Structural basis of RNA polymerase III transcription initiation.
Nature 553,301–306 (2018).
27. Girbig, M. et al. Cryo-EM structures of human RNA polymerase III in
its unbound and transcribing states. Nat. Struct. Mol. Biol. 28,
210–219 (2021).
28. Li, L. et al. Structure of human RNA polymerase III elongation
complex. Cell Res. 31,791–800 (2021).
29. Wang, Q. et al. Structural insights into transcriptional regulation of
human RNA polymerase III. Nat.Struct.Mol.Biol.28,220–227 (2021).
30. Cheung,A.C.M.&Cramer,P.StructuralbasisofRNApolymeraseII
backtracking, arrest and reactivation. Nature 471,249–253 (2011).
31. Ruan, W., Lehmann, E., Thomm, M., Kostrewa, D. & Cramer, P.
Evolution of two modes of intrinsic RNA polymerase transcript
cleavage. J. Biol. Chem. 286,18701–18707 (2011).
Article https://doi.org/10.1038/s41467-023-37109-4
Nature Communications | (2023) 14:1729 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved
32. Sosunov, V. et al. Unified two-metal mechanism of RNA synthesis
and degradation by RNA polymerase. EMBO J. 22,
2234–2244 (2003).
33. Arimbasseri, A. G. & Maraia, R. J. Distinguishing core and holoen-
zyme mechanisms of transcription termination by RNA polymerase
III. Mol. Cell Biol. 33,1571–1581 (2013).
34. Abdelkareem, M. et al. Structural basis of transcription: RNA poly-
merase backtracking and its reactivation. Mol. Cell 75,
298–309.e4 (2019).
35. Vorländer,M.K.,Khatter,H.,Wetzel,R.,Hagen,W.J.H.&Müller,C.
W. Molecular mechanism of promoter opening by RNA polymerase
III. Nature 553,295–300 (2018).
36. Wu, C.-C., Lin, Y.-C. & Chen, H.-T. The TFIIF-like Rpc37/53 dimer lies
at the center of a protein network to connect TFIIIC, Bdp1, and the
RNA polymerase III active center. Mol. Cell Biol. 31,
2715–2728 (2011).
37. Vorländer, M. K. et al. Structural basis for RNA polymerase III tran-
scription repression by Maf1. Nat. Struct. Mol. Biol. 27,
229–232 (2020).
38. Han, Y., Yan, C., Fishbain, S., Ivanov, I. & He, Y. Structural visuali-
zation of RNA polymerase III transcription machineries. Cell Discov.
4,40(2018).
39. Nagarajavel,V.,Iben,J.R.,Howard,B.H.,Maraia,R.J.&Clark,D.J.
Global ‘bootprinting’reveals the elastic architecture of the yeast
TFIIIB-TFIIIC transcription complex in vivo. Nucleic Acids Resv 41,
8135–8143 (2013).
40. Fernández-Tornero, C. et al. Crystal structure of the 14-subunit RNA
polymerase I. Nature 502,644–649 (2013).
41. Fernández-Tornero, C. RNA polymerase I activation and hiberna-
tion: unique mechanisms for unique genes. Transcription 9,
248–254 (2018).
42. Torreira, E. et al. The dynamic assembly of distinct RNA polymerase
I complexes modulates rDNA transcription. Elife 6, e20832 (2017).
43. Maertens, G. N., Engelman, A. N. & Cherepanov, P. Structure and
function of retroviral integrase. Nat. Rev. Microbiol. 20,
20–34 (2022).
44. Abascal-Palacios, G., Jochem, L., Pla-Prats, C., Beuron, F. & Vannini,
A. Structural basis of Ty3 retrotransposon integration at RNA Poly-
merase III-transcribed genes. Nat. Commun. 12, 6992 (2021).
45. Bolton, E. C. & Boeke, J. D. Transcriptional interactions between
yeast tRNA genes, flanking genes and Ty elements: a genomic point
of view. Genome Res. 13,254–263 (2003).
46. Sanz-Murillo, M. et al. Structural basis of RNA polymerase I stalling
at UV light-induced DNA damage. Proc.NatlAcad.Sci.USA115,
8972–8977 (2018).
47. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-
induced motion for improved cryo-electron microscopy. Nat.
Methods 14, 331–332 (2017).
48. Zivanov, J. et al. New tools for automated high-resolution cryo-EM
structure determination in RELION-3. eLife 7, e42166 (2018).
49. Zhang, K. Gctf: Real-time CTF determination and correction. J.
Struct. Biol. 193,1–12 (2016).
50. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to
beam-induced motion correction in cryo-EM single-particle analy-
sis. IUCrJ 6,5–17 (2019).
51. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus
estimation from electron micrographs. J. Struct. Biol. 192,
216–221 (2015).
52. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoS-
PARC: algorithms for rapid unsupervised cryo-EM structure deter-
mination. Nat. Methods 14,290–296 (2017).
53. Pettersen,E.F.etal.UCSFChimera—a visualization system for
exploratory research and analysis. J. Comput. Chem. 25,
1605–1612 (2004).
54. Kelley,L.A.,Mezulis,S.,Yates,C.M.,Wass,M.N.&Sternberg,M.J.
E. The Phyre2 web portal for protein modeling, prediction and
analysis. Nat. Protoc. 10,845–858 (2015).
55. Emsley,P.,Lohkamp,B.,Scott,W.G.&Cowtan,K.Featuresand
development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66,
486–501 (2010).
56. Liebschner, D. et al. Macromolecular structure determination using
X-rays, neutrons and electrons: recent developments in Phenix.
Acta Cryst. D. 75,861–877 (2019).
57. Barkova, A., Asif-Laidin, A. & Lesage, P. Genome-wide mapping of
yeast retrotransposon integration target sites. Methods Enzymol.
612,197–223 (2018).
Acknowledgements
We thank Tornero Laboratory members Federico M. Ruiz for helpful
advice and Srdja Drakulic for technical assistance, and Amandine Bon-
net from Lesage Laboratory for C11 strain and plasmid constructions. We
are grateful to Ernesto Arias-Palomo at CIB-CSIC, Alistair Siebert and
Vinod Vogirala at the Diamond Light Source, and Teresa Bueno and
Rocío Arranz at the CNB-CSIC Cryo-EM Facility in the context of the
CRIOMECORR project (ESFRI-2019-01-CSIC-16) for support in cryo-EM
data collection. We also acknowledge Rafael Núñez at the CIB-CSIC EM
Facility for help during cryo-EM grid preparation, Javier Méndez Viera for
yeast fermentation, and Hung-Ta Chen for providing pCW4 and pCWA-
mut plasmids. This work was supported by the Agence Nationale de la
Recherche (ANR-17-CE11-0025 to P.L., J.A., J.R. and C.F.-T.), the Spanish
Ministry of Science/Agencia Estatal de Investigación (BFU2017-87397-P,
RED2018-102467-T and PID2020-116722GB-I00 to C.F.-T.; PRE2018-
087012 to A.P.-P.), intramural funding from CSIC (2020AEP152 and PIE-
202120E047-Conexiones-Life to C. F.-T.), Fondation pour la Recherche
Médicale (FRM-EQU202203014635 to P.L.; FRM-SPF20170938755 to
A.A.-L.; FRM-ECO202206015504 to B.C.), and intramural funding from
CNRS, Université Paris Cité and INSERM (to P.L.).
Author contributions
P.Q.N. and S.H. prepared Pol III complexes with IN1 and DNA, conducted
biochemical analysis of purified complexes and, together with A.P.-P.,
performed cryo-EM analyses. A.A.-L. and B.C. prepared mutant yeast
strains with the technical help of N.P. and conducted transposition and
integration assays. During article revision, B.C. performed in vivo
experiments while C.C. performed ChIP experiments. J.A., J.R., P.L. and
C.F.-T. conceived and supervised the work. P.L. and C.F.-T. wrote the
paper with input from all authors.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-023-37109-4.
Correspondence and requests for materials should be addressed to
Pascale Lesage or Carlos Fernández-Tornero.
Peer review information Nature Communications thanks Hideki Aihara
and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work.
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