20-nt duplex-forming segment; (ii) the tem-
plate DNA strand comprises a 20-nt duplex-
forming segment followed by a 13-nt tail; (iii)
the Pol IV RNA strand comprises a 9-nt seg-
ment complimentary to the template DNA
strand as well as a 7-nt 5′tail and a 23-nt 3′
tail; (iv) the RDR2 RNA strand comprises a
4-nt primer complimentary to the Pol IV RNA
strand (Fig. 4A). The reconstituted Pol IV–RDR2
bTEC is active in extending the 4-nt RDR2–RNA
primer (fig. S1F). The structure of the com-
plex was determined at a nominal resolution
of 3.16 Å (fig. S3). The cryo-EM map reveals
strong, sharp signals for the 9-base pair (bp)
RNA-DNA hybrid (−9 to−1) at the active center
cleft of Pol IV, and a 4-bp dsRNA (+20 to +23)
in the active center cleft of RDR2, allowing
assignment of the nucleotide register in Pol
IV–RDR2 channels. The cryo-EM map also re-
veals a less strong but unambiguous signal for
dsDNA in the downstream dsDNA channel of
Pol IV and for single-stranded RNA (ssRNA)
in the interpolymerase RNA channel of Pol IV-
RDR2. No signal was observed for Pol IV RNA
in the Pol IV–RNA exit channel (Fig. 4, B to E,
and movie S4).The conformational states of key structural
elements, as well as the interactions of Pol IV
and RDR2, are identical between the struc-
tures of Pol IV–RDR2 bTEC and Pol IV–RDR2
holoenzyme (0.86 RMSD for all Caatoms).
Engagement of the nucleic acid scaffold does
not induce clamp closure of Pol IV as seen in
DNA-bound Pol II (Fig. 4F). The bridge helix
of Pol IV remains in the kinked conforma-
tion, as seen in the structure of Pol IV–RDR2
holoenzyme (Fig. 5A; fig. S4, E, F, and J; and
fig. S9C). The rudder and lid loops remain
disordered, resulting in fully exposed DNASCIENCEscience.org 24 DECEMBER 2021•VOL 374 ISSUE 6575 1583
ACDEFBFig. 4. The cryo-EM structure of Pol IVÐRDR2 bTEC.(A) The nuclei acid
scaffold for structure determination. Pol IV–RDR2 residues are linked to their
contact nucleotides. (Conserved residues between Pol IV and Pol II and
conserved residues of RDR2 in various plant species are colored in blue;
nonconserved residues are colored in black; D1, D2, and R in the parenthesis
represent NRPD1, NRPD2, and RDR2, respectively). Single-letter abbreviations
for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,
Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;
S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) The unsharpened cryo-EM map of
the nucleic acid scaffold. The blue mesh shows the sharpened map for
nucleotides in the active centers of Pol IV and RDR2. (C) The cryo-EM map of
Pol IV–RDR2 bTEC in top and side view orientations. (D) The structure model in
cartoon presentation of Pol IV–RDR2 bTEC. A transparent map of the nucleic
acid scaffold is also shown. (E) Cryo-EM map for the nucleic acid scaffold in
Pol IV–RDR2 channels. (F) (Left) Superimposition between Pol IV–RDR2 bTEC
(bPol IV, colored as above) and Pol II backtracked TEC (bPol II; black); (right)
superimposition between Pol IV–RDR2 bTEC (bPol IV, colored as above) and Pol IV–
RDR2 holoenzyme (gray).RESEARCH | RESEARCH ARTICLES