Science - USA (2021-12-24)

Additionally, the catalytic domain of RDR2
makes extensive contact with the relocated
NRPB9 zinc ribbon (Fig. 3B and movie S3).
Protein sequence alignment analysis sug-
gests that the interface residues on NRPD1 FH
are conserved in Pol IV and distinct from those
of Pol II, conferring the specificity of RDR2
to Pol IV (fig. S6) ( 18 ). The reciprocal interface
residues on RDR2 are conserved in RDR2 ho-
mologs but not in RDR2 paralogs (i.e., RDR1
and RDR6 inA. thaliana), thus supporting a
characteristic role of RDR2 in the Pol IV–
RdDM pathway (fig. S8) ( 13 , 18 ).

An interpolymerase RNA channel bridges
the active sites of Pol IV and RDR2

The Pol IV–RDR2 structure shows that the
active sites of the two RNAPs are directly
connected. The secondary channel of Pol IV
is extended by the dock and guide domains
of RDR2 that form an RNA entry channel
leading to the active site of RDR2 (Fig. 3, E to
G). The secondary channel of Pol IV and the
RNA entry channel of RDR2 thereby form a
continuous half-closed interpolymerase RNA
channel connecting the active site of Pol IV,
where Pol IV–generated RNA (Pol IV RNA) is
synthesized, and the active site of RDR2 where
the RDR2-generated RNA (RDR2 RNA) is syn-

thesized using Pol IV RNA as the template

(Fig. 3H). The secondary channel of msDdRPs

primarily serves as the entrance for substrate

NTP into the active site but also accommo-

dates RNA at the backtracked state (a state in

which msDdRPs move backward on the DNA

template), which is induced under certain cir-

cumstances such as NTP misincorporation,

transcription pausing, and DNA damage ( 40 ).

The presence of an interpolymerase RNA

channel in the Pol IV–RDR2 complex impli-

cates a possible backtracking-triggered RNA

channeling model for dsRNA synthesis by

Pol IV–RDR2, in which the single-stranded

Pol IV RNA is directly delivered to RDR2 through

the interpolymerase RNA channel triggered

by Pol IV backtracking. The 3′terminus of the

Pol IV RNA strand is displaced from the Pol IV

active site, further threaded through the inter-

polymerase RNA channel to the active site of

RDR2, and then directly used as the tem-

plate for synthesis of the RDR2 RNA strand

of dsRNA. This method of RNA delivery is en-

ergetically favorable as the backtracked Pol IV

RNA gains more electrostatic attraction inter-

actions en route to the RDR2 active site through

the interpolymerase RNA channel (Fig. 3H).

Such a mechanism predicts that initiation of

RDR2 RNA synthesis does not require previ-

ous termination and release of single-stranded

Pol IV RNA from the Pol IV RNA exit channel,

thereby preventing Pol IV RNA from degra-

dation by multiple cellular RNA nucleases,

conferring the substrate specificity of RDR2

by directly receiving the Pol IV RNA template

from inside the complex, and ensuring that

dsRNA production is restricted to RdDM loci

of genomic DNA. Three experimental results sup-

port the backtracking-triggered RNA channeling

model: First, Pol IV–RDR2 produces RDR2 RNA

much more efficiently on a backtracking-prone

3 ′-tailed double-stranded DNA template than

on a single-stranded DNA template (fig. S11A)

( 36 , 41 ). Second, the Pol IV–RDR2 complex pro-

tects a much longer region of backtracked RNA

than Pol II (fig. S11B). Third, Pol IV–RDR2 ex-

tends the RDR2–RNA primer only when back-

tracked Pol IV RNA is≥23nt(fig.S11C).The

backtracking-triggered RNA channeling model

has also been proposed in a recent study ( 42 ).

Backtracked Pol IV RNA is threaded into the

RDR2 active site for synthesis of RDR2 RNA

To further demonstrate the backtracking-

triggered RNA channeling model, we recon-

stituted a Pol IV–RDR2 bTEC with a synthetic

nucleic acid scaffold comprising four strands:

(i) The nontemplate DNA strand comprises a

1582 24 DECEMBER 2021•VOL 374 ISSUE 6575 science.orgSCIENCE

5

4

3

1

2

PPoI IVoI IV

RRDR2DR 2

6

MMgg^2 2+PP+

SSlablab

GGuideuide

NNRPD1 FHRPD 1 FH

FF442 442

YY441 441 RR207^207

RR48 48 KK52^52

‘‘W’ loopW’ loop

‘‘YF’ loopYF’ loop

‘‘R’ loopR’ loop

DDockock

WW51 51

RRDR2 dockDR 2 dock

RRDR2 guideDR 2 guide

RRDR2 slabDR 2 slab

NNRPD1 FHRPD 1 FH

RRDR2 catalyticDR 2 catalytic

NRPB9

Zinc ribbon

NNRPB3RPB 3

lloopoop

d domainomain

NNRPB8RPB 8

ββ b barrelarrel

d domainomain

NNRPD1RPD 1

JJawaw

MMgg((R)^2 2+R+)

SSlablab

NRPB9

‘‘YF’ loopYF’ loop Zinc ribbon

YY441 441

FF442 442

FFH stemH stem

FFHTHT

3 secondary channelsecondary channel

4 RNA entry channelRNA entry channel

+

=

PPoI IV/RDR2 channelsoI IV/RDR 2 channels

RRDR2 channelsDR 2 channels

5 RDR2 dsRNA channel

6 NTP entry channel

1 downstream dsDNA channeldownstream dsDNA channel

2 DDNA-RNA channelNA-RNA channel

PPol IV channelsol IV channels

PPoI IVoI IV

RRDR2DR 2

5

4

3

1

2 MMgg

2 2++PP

MMgg((P)^2 2+P+)

NNRPD1RPD 1

ssecondaryecondary

cchannelhannel

RRNA entryNA entry

cchannelhannel

NNRPB8RPB 8

NNRPD2RPD 2

RRDR2DR 2

MMgg((R)^2 2+R+)

DDockock

GGuideuide

SSlablab NNeckeck

ssecondaryecondary

cchannelhannel

RRNA entryNA entry

cchannelhannel

6090

CD

NRPB9

Jaw

NRPB8

β barrel domain

NRPD1

NRPB9

Zinc ribbon

RRDR2DR 2

NRPB3

loop domain

PoI IV

NRPB10

MMgg((R)^2 2+R+)

MMgg((P)^2 2+P+)

NRPD2

1 102 193217278 494 644 725 973 1133

At RDR2 Dock Guide DockGuide Slab Catalytic Neck NeckHead

957

Catalytic

AB

NRPB8

MMgg((R)R^2 2++)

NRPB3

NRPB10 NRPB11

RRDR2DR 2

NRPD2

EG

H

F

6

NRPB9

Zinc ribbon

MMgg((R)^2 2+R+)

in

te

r-

po

ly

m

er

as

e

Rinter-polymerase

NA

c

ha

nn

el

RNA channel

iinter-polymerasenter-polymerase

RRNA channelNA channel

in

te

r-

po

ly

m

er

as

e

Rinter-polymerase

N

A c

ha

nn

el

RNA channel

iinter-polymerasenter-polymeras e

RRNA channelNA channel

MMgg^2 2+RR+

MMgg^2 2++RR

Fig. 3. RDR2 rides on the saddle-like surface of Pol IV.(A) Sketch of the
Pol IV–RDR2 interaction. (B) The interface of RDR2 and Pol IV. Pol IV is shown as
cartoon and half-transparent surface; RDR2 is shown as cartoon. FH, funnel
helices. (CandD) Detailed interaction between RDR2 (cartoon) and Pol IV
(surface). Blue mesh, cryo-EM map. (E) Sketch of the interpolymerase
RNA channel in the Pol IV–RDR2 complex. The circle with a center dot shows
the secondary channel of Pol IV perpendicular to the paper plane. The black
lines delineate the path of the RDR2 RNA entry channel. The red dashed

line shows the boundaries of the RDR2 RNA entry channel and the Pol IV

secondary channel. (F) A scaled-up view of the interpolymerase RNA channel.

(G) An intersection showing the Pol IV secondary channel. (H) Cryo-EM map

(left) and electrostatic surfaces (right) (blue, positively charged; red, negatively

charged) of Pol IV-RDR2 (certain domains omitted for clarity) showing nucleic

acid channels. Channels are delineated by black dotted lines, and channel

boundaries are indicated by red lines. Red dots, catalytic Mg2+of Pol IV (Mg2+(P))

and of RDR2 (Mg2+(R)).

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