and RNA in the active center cleft (Fig. 5A
and figs. S4F and S9B). By contrast, the rud-
der and lid loops in structures of the normal
and backtracked Pol II elongation complexes
exhibit confined conformation that secures
and maintains the transcription bubble, and
thereby confers high processivity to Pol II (fig.
S4G) ( 43 ). Pol IV interacts loosely with dsDNA
in the downstream dsDNA channel (Fig. 5B,
fig. S9E, and fig. S10, D and E), likely due in
part to a lack of dsDNA grippers at the channel
sidewalls (the rudder, clamp head, and lobe
loops) used by Pol II to grip the phosphate
backbones of dsDNA (figs. S6 and S9E) ( 44 ).
In summary, our structures reveal that key
structural elements in the active center cleftadopt conformations distinct from those of
Pol II, and that the distinct conformations re-
main unchanged upon engagement of the back-
tracked nucleic acid scaffold.
The Pol IV RNA (nucleotides of +1 to +24) is
threaded through the interpolymerase RNA
channel from the Pol IV active site into the
RDR2 active site (Fig. 4, B and E, and movie
S4). The channel proximally accommodates
17-nt ssRNA (+2 to +18). The polar residues
(K705, R709, D710, Y716, N736, and K739 of
Pol IV NRPD1; R713 of Pol IV NRPD2; and
R48, K52, R54, Y92, N431, K935, and R937 of
RDR2) in the interpolymerase RNA channel
likely delineate the path of backtracked RNA
by allowing potential H-bond and salt-bridge
interactions with its phosphate backbone (Figs.
4Aand5C,andfig.S10,FandG).RDR2accom-
modates the 4-bp A-form dsRNA at its active
center cleft in a post-translocation state (Fig. 4,
A and B, Fig. 5D, fig. S10H, and movie S5). The
3 ′-OH group of the nucleotide A+20 of RDR2
RNA makes a coordinate bond with the cat-
alytic Mg2+constrained by the catalytic D-triad
ofRDR2(D830,D832,andD834;Fig.5D).The
template nucleotide at the A′site of RDR2 (i.e.,
U+19 of Pol IV RNA) is unpaired, awaiting its
cognate NTP that likely diffuses into the active
site through a shallow NTP channel (Fig. 4A and
Fig. 5, D to F). The RDR2 head domain stabilizes
short dsRNA from the top of the dsRNA groove
but blocks the path of nascent RNA, suggesting
that it must be displaced upon further RNA
extension (Fig. 5D). We propose that the head
module is functionally analogous to domain
s3.2of bacteria-initiatingsfactor or the finger
domain of eukaryotic initiation factor TFIIB,
which initially preorganize the template strand
for efficient initiation of RNA synthesis but are
displaced after RNA extends to 4 to 7 nt form-
ing a stable RNA-DNA hybrid ( 45 , 46 ).Disrupting the Pol IVÐRDR2 interface causes
defects in DNA methylation
On the basis of our structures, we propose that
the Pol IV–RDR2 interaction is a prerequisite
for synthesis of dsRNA precursors ( 20 ). To val-
idate this hypothesis, we determined whether
disruption of Pol IV–RDR2 interactions would
affect RdDM readouts. We generated transgene
lines in which the FLAG-tagged, wild-type
NRPD1, the NRPD1 (DFHT) mutant-bearing de-
letion of the nonconserved tip of NRPD1 FH, and
the NRPD1 (DFHT; M5)-bearing deletion of the
nonconserved tip of NRPD1 FH and the alanine
substitution of five residues at the Pol IV–RDR2
interface (fig. S5D) were expressed from its native
promoter in thenrpd1mutant. The wild-type,
nprd1mutant, and resultant two transgenic
lines were then subjected to bisulfite sequencing
and small RNA sequencing. PNRPD1::NRPD1fully
rescued defects in the RdDM of thenrpd1
mutant; PNRPD1::NRPD1(DFHT) slightly re-
duced the 24-nt siRNA production and DNA1584 24 DECEMBER 2021•VOL 374 ISSUE 6575 science.orgSCIENCE
PPol IIol II
cclamp headlamp headBHBHPol lV SW1rrudderudder
PPol lVol lVPPol lV lidol lV lidNNRPD5 JawRPD 5 JawNNRPD2 lobeRPD 2 lobeTNTRRNANAddownstreamownstream
ddsDNAsDNABHBHTLTLFL1TTbbacktrackedacktracked
PPol IV RNAol IV RNADDNA-RNANA-RNA
hhybridybridMg2+(P)rrudderudder
llididNNRPD2RPD 2P siteA site+1PPoI IV RNAoI IV RNARRDR2DR 2
HHeadeadBHBH
TLTLRRDR2 RNADR 2 RNADD loop loop
NNTPTP
cchannelhannel
RRDR2DR 2
ccatalyticatalyticMg^2 2+((R)R+)P
siteA
siteDDPBBPBB+19RR937 937NN431 431RR54 54KK52 52K705K (^7) RR709 (^07509) Y716Y 716
KK739 739
RR713 713
RRDR2DR 2
NNRPD1RPD 1
PPol IVol IV
Mg2+(P)
MgMg^2 2+((R)R+)
KK935 935
B
RRDR2DR 2
ccatalyticatalytic
NNTPTP
cchannelhannel
RRDR2 RNADR 2 RNA
RRDR2DR 2
ccatalyticatalytic
PPol IV RNAol IV RNA
MgMg^2 (R)2+(R+) NNTPTP
cchannelhannel
E
C
A
D
F
KN736 736
RD710 710
RR48 48
YY92 92
MgMg^2 2+((R)R+)
NNRPD1RPD 1
bbacktrackedacktracked
PPoI IV RNAoI IV RNA
NTNT
Fig. 5. Detailed interaction between Pol IV-RDR2 and the nucleic acid scaffold.(A) The RNA-DNA
hybrid in the Pol IV active center cleft. Blue rectangles highlight the product site (P site) and nucleotide
insertion site (A site) of the Pol IV active center. (B) Interaction between Pol IV and downstream dsDNA.
Red dashes indicate the position of Pol II clamp-head domain. Gray dashes represent disordered the lid and
rudder loops. (C) The backtracked Pol IV RNA makes interaction with polar residues (shown as purple and
gray dots) in the interpolymerase RNA channel. The dashed line indicates the interface of Pol IV and RDR2.
(D) RDR2 active center. Blue rectangles highlight the product site (P′site) and nucleotide insertion site (A′
site) of the RDR2 active center. DPBB, conserved double-psib-barrel domain of msRNAPs. (E) Illustration and
(F) cryo-EM map of RDR2 showing the NTP channel.
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