Science - USA (2021-12-24)

loop, the lid loop, the bridge helix, and the
trigger loop) compared with those of Pol II.

Pol IV lacks surfaces for recruiting Pol II
transcription factors

Aside from the key motifs inside the active
center cleft, Pol IV shows greater sequence
and conformational differences on its surface
compared with Pol II, as it lacks anchoring
surfaces for Pol II initiation and elongation
factors. For example, TFIIB interacts with con-
served residues of multiple surface patches
on Pol II (fig. S7). By contrast, all of the surface
patches used for recruiting TFIIB are either
absent or with nonconsensus residues in Pol IV

(Fig. 2D and fig. S7). These structural features

explain previous observations that TFIIB,

TFIIF, and other Pol II transcription initia-

tion factors were not detected in the LC-MS/MS

analysis of the immunoprecipitated Pol IV ( 16 ).

Because TFIIB is the central hub for docking

of TBP/TFIID-bound promoter DNA to Pol II

( 33 , 34 ), the loss of TFIIB-anchoring surfaces

on Pol IV supports the idea that Pol IV does not

recognize Pol II promoters and uses a mech-

anism for transcription initiation distinct from

that of Pol II ( 21 – 24 ).

The Pol IV–RDR2 holoenzyme structure

also suggests that Pol IV is defective in binding

to TFIIS, the proofreading elongation factor

of Pol II that plays a key role in maintaining

transcription fidelity and resolving backtrack-

ing ( 35 ). TFIIS is an extended protein with two

domains (domain II and III) connected by an

interdomain linker. Domain II docks on the

Pol II–RPB1 jaw domain, and the fully ex-

tended linker helps domain III insert into the

active site through the secondary channel

of Pol II (fig. S4L) ( 36 , 37 ). Pol IV does not

have the anchor helix on the NRPD1 jaw do-

main for anchoring domain II of TFIIS (Fig. 2E)

( 38 ). The entry route of the TFIIS interdomain

linker is occupied by the relocated NRPB9 zinc

ribbon of Pol IV (Fig. 2E and fig. S4, K and L).

The sequence and structure features of Pol IV

1580 24 DECEMBER 2021•VOL 374 ISSUE 6575 science.orgSCIENCE

MMainain

clefcleftt

RRDR2DR 2

NNRPB9RPB 9

JJawaw

NNRPB12RPB 12

NNRPB6aRPB 6 a

NNRPD5RPD 5

NNRPD4RPD 4

NNRPD7RPD 7

NNRPD1RPD 1

NNRPB3RPB 3 NRPD2NRPD^2

ssecondaryecondary

cchannehannell

B

90 NNRPD1RPD^1 RRDR2DR^2

NNRPD5RPD 5

NNRPD2RPD 2

NNRPD7RPD 7

NNRPD4RPD 4

NNRPB9RPB 9

JJawaw

NNRPB9RPB 9

NRPB10NRPB 10 ZZinc ribboninc ribbon

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

At NRPD7 At NRPB8b At NRPB9a At NRPB10 At NRPB11 At NRPB12

1 174 1 63 96 146 1 38 53 114 1 71 1 116 1 70

ββ--barrelbarrel looploopββ-barrel-barrel JawJawLinkerribbonrZinciZbibnocn

At NRPB3a

1 319

At NRPD4 At NRPD5a At NRPB6a

1 205 1 135 205 1 52 144

looploop TTailail AssemblyAssembly

190 265

1 85 218 319 348 401 474 621 805 954 1079 1215 1276 1453

At NRPD1 CClamplamp

ccoreore DDockock Pore 1Pore^1 FunnelFunnel JawJaw CCleftleft

CClamplamp

ccoreore

CClamplamp

hheadead DDeCL-likeeCL-like

CClamplamp

CCleftleft corecore

837 877 911

Active siteActive site BH(P) TL(P)

MBS

1 49 223 399 460 542 627 698 801 907 10721096 1172

At NRPD2 Ex1Ex 1 PProtrusionrotrusion LLobeobe ProtrusionProtrusionForkFork Ex2Ex 2 Ex1Ex (^1) bindingbHHybridiynbridding ClClampamp
An
ch
o
Ancho
HHybridybrid rr
WallWall bbindinginding
At PoI IV
1 102 193217 278 494 644 725 935973 1133
At RDR2 DDockock GuideGuide oDkDockcGuideGuide SSlablab CatalyticCatalytic NNeckeck ekNNeckc HHeadead
MBS 867900906957
CCatalyticatalytic
A
JJawaw
MMainain
clefcleftt
RRDR2DR 2
NNRPB9RPB 9
JJawaw
NNRPB12RPB 12
NNRPB6aRPB 6 a
NNRPD1RPD 1 NRPD5NRPD^5
NNRPB3RPB 3 NRPD2NRPD^2
C
90
NNRPD1RPD 1
RRDR2DR 2
NNRPD5RPD 5
NNRPD2RPD 2
NNRPB9RPB 9
JJawaw
NNRPB9RPB 9
NRPB10NRPB 10 ZZinc ribboninc ribbon
MMgg^2 2+((P)P+)
MMgg((R)^2 2+R+)
MMgg((R)^2 2+R+)
MMgg((P)^2 2+P+)
NNRPD2RPD 2 NNRPD1RPD 1 RRDR2DR 2
NNRPD5RPD 5
NNRPB8RPB 8
NNRPB11RPB 11
NNRPB12RPB 12
NNRPB10RPB 10
NNRPB3RPB 3
NNRPB9RPB 9
JJawaw
NNRPB9RPB 9
ZZinc ribboninc ribbon
90
NNRPD2RPD 2 NNRPD1RPD 1 RRDR2DR 2
NNRPD5RPD 5
NNRPB8RPB 8
NNRPB11RPB 11
NNRPB12RPB 12
NNRPB10RPB 10
NNRPB3RPB 3
NNRPB9RPB 9
JJawaw
NNRPB9RPB 9
ZZinc ribboninc ribbon
90
MMgg^2 2+((R)R+)
MMgg((P)^2 2+P+)
BH(R) TL(R)
ssecondaryecondary
cchannehannell
Fig. 1. The cryo-EM structure of theA. thalianaPol IVÐRDR2 holoenzyme.
(A) A schematic presentation of Pol IV–RDR2 subunits. MBS, Mg2+binding site;
BH(P), Pol IV bridge helix; TL(P), Pol IV trigger loop; BH(R), RDR2 bridge helix;
TL(R), RDR2 trigger loop. (B) Cryo-EM map of the Pol IV–RDR2 holoenzyme.
(C) Structure model of the Pol IV–RDR2 holoenzyme. Subunits are colored
as in (A). Red dashed circle shows the Pol IV secondary channel. Black dashed
lines indicate the main cleft of pol IV. Red dots indicate catalytic Mg2+in the Pol
IV (Mg2+(P)) and RDR2 (Mg2+(R)) active centers.
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