Science - USA (2021-11-12)

that Integrator binding is inconsistent with
EC formation (fig. S8). Integrator excludes
PAF binding because the N-terminal region of
INTS1 clashes with PAF subunits PAF1 and
LEO1 (fig. S8A) and INTS2 clashes with the
PAF subunit CTR9 (fig. S8B). Integrator also
excludes SPT6 because INTS11 and INTS6
clash with the SPT6 core and tandem SH2
(tSH2) domain, respectively (fig. S8C). Thus,
Integrator interferes with EC formation not
only by counteracting phosphorylation but
also by steric hinderance.
Finally, our structure provides clues as to
how Integrator may facilitate Pol II termi-
nation. Termination requires the release of
nucleic acids from the Pol II active center cleft,
particularly release of the DNA-RNA hybrid
that is encircled within Pol II by the closed
clamp ( 38 ) and by the DNA clamp of DSIF
that consists of the SPT5 NGN domain and
SPT4 ( 23 ). Our structure shows that the Pol II
clamp is still closed but not restricted by Inte-

grator and that the DSIF DNA clamp is open

because the NGN domain and SPT4 are mo-

bile (Fig. 1C). DSIF also forms a clamp on ex-

iting RNA with its SPT5 KOWx-4 domain ( 23 ),

which is bound by the INTS11 endonuclease.

We speculate that RNA cleavage facilitates

opening of the RNA clamp of DSIF and ex-

poses an uncapped RNA 5′end that is a sub-

strate for the XRN2 nuclease that can cause

termination ( 39 ).

In conclusion, our PEC-Integrator-PP2A struc-

ture represents a pretermination complex

and provides insights into the mechanisms of

Integrator-mediated transcription regulation

(fig. S11D). Integrator recognizes the PEC by

forming contacts with Pol II, DSIF, and NELF;

positions the PP2A phosphatase to counteract

PEC phosphorylation; sterically interferes with

the association of the positive elongation fac-

tors SPT6 and PAF; and positions its cleavage

module to cleave nascent RNA ~20 nt from

the Pol II active site. Finally, Integrator induces

a PEC conformation that is consistent with the

release of nucleic acids and transcription ter-

mination. These mechanisms likely underlie

not only transcription attenuation in the 5′re-

gion of protein-coding genes but also termina-

tion at the 3′end of noncoding transcription

units, including enhancers.

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ACKNOWLEDGMENTS

We thank U. Steuerwald for support at the microscope and for

maintaining the EM facility, T. Schulz for maintaining cells

and thymus, P. Rus and U. Neef for running the insect cell

facility, and F. Wagner for providing the continuous carbon used

in cryo-grid preparation.Funding:P.C. was supported by the

Deutsche Forschungsgemeinschaft (EXC 2067/1-390729940)

and the European Research Council (Advanced Investigator

Grant CHROMATRANS 882357). H.U. was supported by the

Deutsche Forschungsgemeinschaft (SFB860).Author

contributions:I.F. designed and performed all experiments and

data evaluation except as follows. Y.C. prepared PP2A and

assisted with structural modeling. C.D. assisted with cryo-EM

886 12 NOVEMBER 2021•VOL 374 ISSUE 6569 science.orgSCIENCE

Linker Proximal CTD Distal CTD with crosslinked Lys residues

RPB1

Distal CTD Lys crosslink sites

PP2A-C active site

Open space to

accommodate CTD

Downstream

DNA

CTD peptide

A

B

C INTS7 D

INTS4

INTS4

INTS 2

INTS6

INTS7

S5

P6

S7 Y1

S2

P3

T4

S5

P6

S7

Y1 S2

R73

R106

K105

P3

INTS7

INTS4

3

28

53

-5.0 kT/e 5.0

D538

E536

D533

D540

D541

INTS 2

INTS 2

K K K K K K K K

400 Å 1400 Å

180°

M1484

M1484

Fig. 4. Integrator-CTD interaction and Pol II dephosphorylation.(A) Schematic showing organization of
the Pol II subunit RPB1. A linker connects the structured region of RPB1 to the CTD, which consists of a
proximal part and a distal part that contains lysine residues (red). The length of a fully extended linker
and CTD are indicated. (B) Location of lysine residues that cross-linked to the distal CTD within an open
space formed between Pol II and Integrator (black outline). (C) Two CTD repeats are bound to the open space
in (B), interacting with the Integrator surface that is colored according to electrostatic surface potential ( 40 ).
Blue and red showing positively and negatively charged areas, respectively. (D) Interactions of the two CTD
repeats with INTS2, INTS4, and INTS7. The observed cryo-EM density is shown as a purple semitransparent
surface. Charged residues in the vicinity of the CTD are shown as sticks.

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