Vous êtes sur la page 1sur 2

news and views

Closed for business: exit-channel coupling to active site


conformation in bacterial RNA polymerase
Craig T Martin & Karsten Theis

npg

2014 Nature America, Inc. All rights reserved.

Fluorescent probes at the exit channel and near the active site of RNA polymerase shed light on how transcriptional
pausing is regulated.
In their cellular contexts, RNA polymerases are
not smoothly running copying machines creating RNA duplicates from archival DNA. It has
been known that these translocating enzymes
undergo sequence- and factor-defined pausing
during the synthesis of the thousands of bases
comprising a transcript and that programmed
pauses are critical for regulatory proteins to
bind the transcription complex and to direct
termination of transcription13. Programmed
pauses during RNA synthesis also regulate a
wide variety of biological processes (Fig. 1),
including cotranscriptional protein synthesis
by a trailing ribosome, RNA processing and
cotranscriptional folding of structured RNAs
such as regulatory riboswitches47.
How is this pausing programmed? Structures
within the elongating bacterial RNA polymerase
that must be maintained include downstream
DNA interactions of the and subunits,
which together form a clamp around the
downstream duplex, an 8- to10-bp RNADNA
hybrid (and its accompanying melted-DNA
bubble), and the path of the emerging 5 end
of the nascent transcript through an RNA-exit
channel (Fig. 2)8,9. With the wealth of structural studies in recent years, we now know that
this basic structure is dynamic, with distinct
structural states associated with various steps
of transcription1016. In particular, the downstream clamp exists in an open state in the
absence of DNA and then closes down around
the DNA duplex within functional initiation
and elongation complexes.
More recently, structures of paused elongation complexes of Thermus thermophilus RNA
polymerase (Fig. 2b) have revealed that the
downstream clamp is reopened in elemental
(or off-pathway) paused elongation complexes,
thus raising interesting questions regarding the
dynamics of the clamp state during elongation17.
Craig T. Martin is at the Department of
Chemistry, University of Massachusetts,
Amherst, Amherst, Massachusetts, USA.
Karsten Theis is at the Department of
Chemical and Physical Sciences,
Westfield State University, Westfield,
Massachusetts, USA.
e-mail: cmartin@chem.umass.edu

Until recently, the RNA-exit channel had


been viewed as a static passageway for singlestranded RNA. However, upon downstream
clamp opening in the elemental paused state,
the RNA-exit channel also widens, as does the
protein environment around the RNADNA
hybrid. In addition, the protein-bridge helix,
positioned near the polymerase active site,
kinks and blocks the +1 template-strand base
from being optimally positioned for catalysis.
A variety of apparently coupled motions occur
simultaneously in the paused state to thwart
nucleotide addition.
In this issue, Landick and colleagues18
directly test proposed mechanistic elements
of the conformational changes. In particular,
they address how formation of an RNA hairpin within the exit channel induces pausing of
transcription and describe its interplay with

other factors that regulate pausing. The authors


use an artificially halted (scaffold-constructed)
elongation complex to specifically model the
pause hairpin of the his operon leader sequence.
They then add a complementary RNA that
forms a duplex with the scaffold RNA within
the channel in trans to mimic formation of the
hairpin19. Incorporation of the fluorescent
base reporter pyrrolo-C into the scaffold RNA
permits duplex formation to be monitored in
real time, because annealing of the antisense
RNA to the target reduces quenching of the
reporter fluorescence, thereby increasing the
observed fluorescence. This powerful approach
allows the direct measurement of kinetic events
in the process20.
Somewhat surprisingly, the added complementary RNA binds its target readily (about
half as fast as free target) even in the closed

RNAP elongating

RNAP pausing

Transcription
versus
translation
Ribosome lagging: Rho binds,
allowing termination

Ribosome blocks Rho access,


preventing termination

Transcription
versus
termination
Rho cannot catch up,
preventing termination

Promotes long-distance
RNA structures

Rho catches up,


allowing termination

Transcription
versus
RNA folding

Favors short-range
RNA structures

Figure 1 Regulated pausing during transcription elongation by RNA polymerase (RNAP) affects multiple
biological processes. The cartoons show RNA (red line) leaving the exit channel of the RNA polymerase
(light blue shape on the right) during active elongation (left) or a pausing event (right). The subunits of the
ribosome are indicated by large and small purple ovals; termination factor Rho is represented as a blue
hexagon; complementary RNA sequences capable of forming secondary structures are indicated by blue,
light and dark green sections, respectively.

nature structural & molecular biology volume 21 number 9 september 2014

741

news and views

Closed (active)

Open (paused)

+1

+1

Mg2+

Mg2+

npg

2014 Nature America, Inc. All rights reserved.

Figure 2 Structural differences between the active elongation complex (left) and the paused elongation
complex (right). To afford a view of the RNA (red) and DNA (blue and cyan), RNA polymerase subunits
(green) and (yellow) are shown in a cut surface representation, with the bridge helix (yellow cylinder)
and the active site magnesium ion (black dot) in front of the slab. Disordered structural elements in the
paused state are sketched in their approximate locations (red dots, RNA; green hatched oval, flap tip).
Asterisks denote the locations of cysteine cross-links used by Landick and colleagues18. Models are based
on PDB 2O5I (elongation complex) and 4GXY (paused elongation complex).

state of the exit channel. This suggests that


the closed channel provides minimal barriers
to the formation of duplexes and presumably
RNA hairpins. Furthermore, the rate of dissociation of the duplex is not altered within the
channel, thus suggesting that the exit channel
minimally stabilizes the hairpin.
Introducing targeted disulfide cross-links
that lock the RNA-exit channel in open or
closed states (Fig. 2) confirms these observations. Although the closed conformation allows
duplex formation, pause-lifetime measurements indicate that the closed conformation
prevents stimulation of pausing by an RNA
duplex in the exit channel. Conversely, locking
the channel in the open state stimulates pausing
even in the absence of an RNA duplex. The
authors conclude that most or all of the pause
stimulation due to formation of an RNA hairpin in the exit channel arises from stabilization
of the open clamp conformation.
Previous studies17,21 have revealed that
clamp motion is coupled to the geometry of
the active site, suggesting a mechanism for the
stimulation of pausing. Consistently with this
proposed coupling, mutations near the active
site that restrict pausing also slow the formation of exit-channel duplexes.
The data thus support the notion of bidirectional regulation via clamp opening and closing,

742

with the active site sensitive to changes


at the exit channel and vice versa. Experiments
with transcriptional regulators that affect
pausing further support this notion: binding of NusG and RfaH (specifically their
N-terminal domains) near the active site
promotes trigger-loop folding and clamp
closure and thus suppresses pausing19,22.
NusA, interacting with the flap domain at
the exit channel, promotes RNA hairpin
mediated exit-channel clamp opening and
thus increases pausing.
Turning to effects on RNA polymerase
translocation, the authors incorporated the
fluorescent base analog 6-MI into either the
DNA template or nontemplate strands to monitor movement of the transcription bubble23.
As the polymerase translocates, a probe at the
melted upstream edge of the bubble reanneals
with its partner, increasing its fluorescence.
Similarly, an annealed probe at the downstream
edge of the duplex is released, decreasing its
fluorescence. Together, these probes enable
the polymerase translocation register to be
followed. The results suggest that formation of
the exit-channel duplex both inhibits forward
translocation and shifts the equilibrium toward
the inactive pretranslocated configuration.
Of course, both of these effects may contribute to the overall pause lifetime of a complex.

Consistently with the established role of the


flap domain, deletion of the flap tip abolishes
these effects.
Although the magnitudes of the individual
changes observed in this study are relatively
small, together they present a largely consistent picture of the role of hairpin formation in
modulating elongation-complex pause
lifetimes. Building on a vast body of work in this
area, the use of fluorescent base analogs to follow
both hairpin formation and translocational states of the polymerase has greatly
enhanced understanding of the subtle
but biologically important effects of the regulated progression of RNA polymerase along its
DNA template.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
1. Chan, C.L., Wang, D. & Landick, R. J. Mol. Biol. 268,
5468 (1997).
2. Hollands, K., Sevostiyanova, A. & Groisman, E.A.
Proc. Natl. Acad. Sci. USA 111, E1999E2007
(2014).
3. Landick, R. Biochem. Soc. Trans. 34, 10621066
(2006).
4. Larson, M.H. et al. Science 344, 10421047
(2014).
5. Lai, D., Proctor, J.R. & Meyer, I.M. RNA 19,
14611473 (2013).
6. McGary, K. & Nudler, E. Curr. Opin. Microbiol. 16,
112117 (2013).
7. Perdrizet, G.A., II, Artsimovitch, I., Furman, R.,
Sosnick, T.R. & Pan, T. Proc. Natl. Acad. Sci. USA
109, 33233328 (2012).
8. Opalka, N. et al. PLoS Biol. 8, e1000483
(2010).
9. Vassylyev, D.G., Vassylyeva, M.N., Perederina, A.,
Tahirov, T.H. & Artsimovitch, I. Nature 448, 157162
(2007).
10. Bae, B. et al. Proc. Natl. Acad. Sci. USA 110,
1977219777 (2013).
11. Murakami, K.S. J. Biol. Chem. 288, 91269134
(2013).
12. Belogurov, G.A. et al. Nature 457, 332335
(2009).
13. Zhang, Y. et al. Science 338, 10761080 (2012).
14. Tagami, S. et al. Nature 468, 978982 (2010).
15. Vassylyev, D.G. et al. Nature
448, 163168
(2007).
16. Tagami, S. et al. Genes Dev.
28, 521531
(2014).
17. Weixlbaumer, A., Leon, K., Landick, R. & Darst, S.A.
Cell 152, 431441 (2013).
18. Hein, P.P. et al. Nat. Struct. Mol. Biol. 21, 794802
(2014).
19. Kolb, K.E., Hein, P.P. & Landick, R. J. Biol. Chem.
289, 11511163 (2014).
20. Liu, C. & Martin, C.T. J. Mol. Biol. 308, 465475
(2001).
21. Nayak, D., Voss, M., Windgassen, T., Mooney, R.A. &
Landick, R. Mol. Cell 50, 882893 (2013).
22. Zhang, J., Palangat, M. & Landick, R. Nat. Struct. Mol.
Biol. 17, 99104 (2010).
23. Hawkins, M.E. Methods Enzymol. 450, 201231
(2008).

volume 21 number 9 september 2014 nature structural & molecular biology