thus imply an incompatible interaction be-
tween TFIIS and Pol IV, consistent with the
absence of TFIIS in previous and our LC-MS/MS
analysis of affinity-purified Pol IV. Our results
suggest that TFIIS is unable to cleave back-
tracked RNA of Pol IV and resolve arrested Pol
IV as it does during Pol II transcription ( 16 ).
RDR2 rides on the saddle-like surface of Pol IV
RDR2 belongs to a large family of cRdRPs and
plays a central role in the RNA interference
(RNAi) pathway in plants, fungi, and metazoans
( 30 , 39 ). Like those of msDdRPs, the active cen-
ter cleft of RDR2 is located at the bottom of
two lobes (Fig. 2G). The slab and guide domains
comprise one lobe, and the neck domain com-
prises the other. The catalytic center of RDR2
is composed of structural elements similar to
the D loop, the bridge helix, and the trigger
loop of msDdRPs (fig. S4H). The structural
similarities imply that RDR2 likely accom-
modates nucleic acids in the active site and
catalyzes the RNA extension in a manner sim-
ilar to that of msDdRP.
RDR2 rides on the saddle-like surface of
Pol IV created by the NRPB3 loop domain,
the NRPB8b-barrel domain, the NRPB9 zinc
ribbon, the NRPD1 cleft, and the NRPD1 FH
(Fig.3,AandB,andmovieS3).RDR2wedges
its dock domain into the Pol IV saddle (Fig. 3B).The conserved residue W51 of the W loop of
the RDR2 dock domain is secured by a hydro-
phobic pocket on NRPD1 FH; the nearby resid-
ues R48 and K52 of the W loop of the RDR2
dock domain make potential salt-bridge inter-
actions with residue D710 of NRPD1 FH (Fig. 3C
and fig. S5A). The guide and slab domains of
RDR2 also insert two loops (the R loop and the
YF loop) into the Pol IV saddle. Residue R207
of the R loop likely makes salt-bridge bonds
with E641 and E642 of NRPD1 FH (Fig. 3C and
fig. S5B); residues Y441 and F442 of the YF
loop insert into hydrophobic pockets on the
interface of the NRPD1 FH and NRPB9 zinc
ribbon (Fig. 3, C and D, fig. S5C, and movie S3).SCIENCEscience.org 24 DECEMBER 2021¥VOL 374 ISSUE 6575 1581
Mg2+ BBHHNNRPD2RPD 2NNRPD1RPD 1NRPD5NRPB9NRPB12PoI IV vs PoI II (PDB: 1WCM)NNRPD7RPD 7
NNRPD4RPD 4NNRPB12RPB 12NNRPB3RPB 3pprotrusionrotrusioncclamplampmain
cleftwwallall
DDockockllobeobeDDockock
SlabSlab GGuideuideNNeckeckCCatalyticatalyticHHeadeadMain
cleftLidB HBHMg2+FL1rrudderudderDownstream
dsDNA channel
DNA-RNA hybrid
channel
RNA
exit
channelT LTLTTFIIBFIIB
PPol IIol II
NNRPD1RPD 1NNRPB11RPB 11D EF GNNRPD2RPD 2NRPB11NRPB8RRDR2DR 2NRPD5
NNRPB9RPB 9
ZZinc ribboninc ribbonNRPB12
NRPB3RRPB9PB 9
ZZinc ribboninc ribbonFunnel
helicesNNRPD1RPD 1RPB9 JawNRPB10HHeadeadSSlablabNNeckeckCCatalyticatalyticMain
cleft9090CA BNNRPD7RPD 7NNRPD4RPD 4NNRPD5RPD 5
cclamplamppprotrusionrotrusionNNRPB12RPB 12NNRPB3RPB 3NNRPB9RPB 9RRDR2DR 2
MMgg^2 2++ BBHHllobeobemain
cleft wallwall JawJaw 9090NNRPB9 JawRPB 9 Jaw
((Pol IV Pol IV ))NRPB9
Zinc ribbon
(Pol IV )Funnel
helicesTFIIS domain III
(Pol ll)NNRPD1RPD 1NRPD1 JawTFIIS domain II
(Pol II)RRDR2DR 2NNRPB9 JawRPB 9 JawNNRPD2RPD 2PoI IV
PoI IIFig. 2. Structural features of Pol IV and RDR2.(A) Cryo-EM map of Pol IV.
Colors as in Fig. 1. (B) Structure superimposition betweenA. thalianaPol IV
[(colored as in (A)] and yeast Pol II (gray; PDB: 1WCM). (C) The lid, rudder, and
fork loop 1 (FL1), bridge helix (BH), and trigger loop (TL) of Pol IV. (D) Pol IV
mutates contact surfaces for TFIIB. The red surface patches highlight mutated
residues of Pol IV on the corresponding TFIIB-contact surface of Pol II. The Yeast
TFIIB (blue) is superimposed onto the structure of Pol IV. (E) TFIIS is sterically
incompatible with Pol IV NRPB9 (cyan). The Pol II–bound TFIIS (PDB: 1Y1V;
blue) is superimposed onto the structure of Pol IV–RDR2 , and a dashed oval
indicates the position of the anchor helix of Pol II. (F) NRPB9 Zinc ribbon
(cyan) is relocated in Pol IV compared with that (gray) in Pol II (PDB: 1WCM).
(G) Cryo-EM map of RDR2.RESEARCH | RESEARCH ARTICLES