Science - USA (2021-11-12)

(Antfer) #1

subunit INTS3 ( 31 , 32 ), which is also mobile
but located nearby according to our cross-
linking data (figs. S5A and S6E).
Close to interface D, PP2A adopts a defined
position with respect to the PEC (Fig. 1B).
INTS6 forms a bridge between NELF and
PP2A (fig. S4F), consistent with the observa-
tion that INTS6 mediates PP2A recruitment
to transcriptional pause sites ( 16 ). PP2A de-
phosphorylates the C-terminal domain (CTD)
of Pol II ( 14 , 16 ). Our structure shows that the


phosphatase active site in subunit PP2A-C is
located ~75 Å from the last ordered residue in
RPB1 (M1484), which marks the beginning of
the flexible linker that connects to the CTD
(Fig. 4, A and B). Therefore, most residues in
the linker and any CTD residue can in prin-
ciple reach PP2A for dephosphorylation.
Integrator also binds to the CTD ( 1 ) and
this can be facilitated by CTD phosphorylation
( 7 , 33 – 35 ). In our cryo-EM density, we ob-
served two heptad repeats of the unphosphoryl-

ated CTD that could be modeled using the two
Y1 residues as anchors (Fig. 4, C and D). The
CTD repeats bind between Pol II and Integra-
tor within an open space that is lined by resi-
dues that form cross-links with the distal part
of the CTD (Fig. 4, A and B). The two CTD
repeats form extensive hydrophobic contacts
with INTS2, INTS4, and INTS7 (Fig. 4, C and
D). Our cryo-EM data show that Integrator
can bind the two CTD heptad repeats without
stably binding to the PEC surface (fig. S11, A to
C). This observation is consistent with Inte-
grator being recruited to transcribing Pol II
through the CTD and may explain the reported
occupancyofgenebodieswithIntegrator
( 2 , 6 , 9 , 11 ).
The observed Integrator-bound CTD con-
formation suggests that phosphorylation of
the two Y1 residues could be accommodated
and that the negatively charged phosphate
groups could bind the conserved positively
charged INTS7 residues R73 and K105/R106
(Fig. 4D). Such an interaction of Integrator
with the Y1-phosphorylated CTD may explain
why Y1 phosphorylation occurs in Pol II that
pauses in the promoter-proximal region and
how it regulates Integrator recruitment and
termination of mammalian Pol II ( 35 ). It was
also reported that CTD phosphorylation at
S7 residues has a role in Integrator recruit-
ment ( 33 , 34 ). Although binding of an S7-
phosphorylated CTD is inconsistent with our
structure, it is possible that a S7-phosphorylated
CTD binds differently to recruit Integrator to
specific genes ( 36 ).
Productive transcription involves the con-
version of the PEC to an active elongation
complex (EC*) containing DSIF, the PAF1 com-
plex (PAF), and SPT6 ( 37 ). Comparison of our
structure with the EC* structure ( 37 ) shows

SCIENCEscience.org 12 NOVEMBER 2021•VOL 374 ISSUE 6569 885


RBP3

A Interface A Interface B

C

Interface D

D

B

RPB2 external domain

INTS1 RPB2 binding domain
INTS7

ß-CASP

MBL

INTS11

NELF-B

INTS6

Top view

Side view

(^1340)
1
15
(^8114)
15
17 16
5
15
6
9
(^910)
Exiting
RNA
A
B KOWx-4
SPT5 Interface C
Fig. 2. Integrator-PEC interfaces.(A) Location and magnified views of interfaces A and B using the top and
side views of the structure, respectively. (B) Location and magnified views of interfaces C and D using the
side view of the structure.
ACB
Active
center
groove
INTS11
-11
-1
Pol II active site metal A INTS11 active site Zn2+^
Exiting
RNA
INTS11 (closed)
~17° ~17°
INTS11 (open)
ß-CASP
MBL
ß-CASP
CPSF73CPSF 73
-16
-17 -20
3’
46 nt
InputInt(wt)Int(E203Q)PEC-Int(wt)PEC-Int(E203Q) Pol II-NELF-Int(wt)Pol II-DSIF-Int(wt)Pol II-Int(wt)
27 nt
25 nt
21 nt
17 nt
12 3 456 7 8
Input RNA
Cleavage products
~20 nt from Pol II
active site
Free
Integrator
Bound
Integrator
Shorter RNA
cleavage products
Fig. 3. Integrator-mediated RNA cleavage.(A) Comparison of our structure of
INTS11 with that of the unbound Integrator-PP2A complex (PDB 7CUN) ( 15 ) indicating
a rotation of theb-CASP domain that results in opening of the active center groove.
The view is the same as in Fig. 2B. (B) Path of nascent RNA from the Pol II active
site to the Integrator endonuclease active site. The register of the RNA nucleotides
from the RNA 3′end in the Pol II active site (register–1) is indicated. RNA nucleotides



  • 17 to–23 are modeled by superposition of RNA bound to CPSF73 (PDB 6V4X) ( 26 ).
    The Pol II active site metal A and the INTS11 active site Zn atoms are shown as
    magenta and dark gray spheres, respectively. (C) Integrator is active in RNA cleavage
    in vitro. Int(wt), wild-type Integrator-PP2A; Int(E203Q), Integrator-PP2A complex
    with INTS11-E203Q mutation. See fig. S10 for the cleavage assay performed with the
    hairpin RNA used for structural analysis and replicates.


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