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EMBO reports 8, 3, 271–278 (2007)
doi:10.1038/sj.embor.7400888
AOP Published online: 9 February 2007
Upstream promoter sequences and  CTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex
Sara Cellai1, Laura Mangiarotti1, Nicola Vannini1, Nikolai Naryshkin2†, Ekaterine Kortkhonjia2‡, Richard H Ebright2 & Claudio Rivetti1
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1 Department of Biochemistry and Molecular Biology, University of Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy
2 Department of Chemistry, Waksman Institute, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, USA
To whom correspondence should be addressed
Richard H Ebright Tel: +1 732 445 5179; Fax: +1 732 445 5312; E-mail: ebright@mbcl.rutgers.edu
Claudio Rivetti Tel: +39 0521 905649; Fax: +39 0521 905151; E-mail: claudio.rivetti@unipr.it
† Present address: PTC Therapeutics, 100 Corporate Court, South Plainfield, New Jersey 07080, USA
‡ Present address: Department of Chemistry and Biochemistry, University of California at Los Angeles (UCLA), 607 Charles E. Young Drive East, Los Angeles, California 90095, USA
Received 26 May 2006; Accepted 23 November 2006; Published online 9 February 2007.
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Abstract
We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP–promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP -subunit carboxy-terminal domain and on the presence and length of the RNAP -subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP–promoter open complex at the  PR promoter is not a general feature of RNAP–promoter open complexes.
EMBO reports 8, 3, 271–278 (2007)
doi:10.1038/sj.embor.7400888
AOP Published online: 9 February 2007
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Introduction
Atomic force microscopy (AFM) allows straightforward detection and quantification of DNA compaction by DNA-binding proteins (Rivetti et al, 1999; Verhoeven et al, 2001; Heddle et al, 2004). DNA compaction is observed as a reduction of the DNA contour length in the presence of the DNA-binding protein of interest with respect to free DNA.
In previous work, using AFM, it has been shown that Escherichia coli RNA polymerase (RNAP) results in massive,  30 nm, apparent DNA compaction on formation of a catalytically competent RNAP–promoter open complex (RPo) at the PR promoter (Rivetti et al, 1999). On the basis of the dimensions of RNAP (10 nm  10 nm 15 nm; Zhang, 1999), the structure of RNAP (Zhang, 1999), and the modelled structure of RPo (Naryshkin et al, 2000), this massive apparent DNA compaction must arise from wrapping or spooling of upstream promoter DNA (promoter positions -100 to -40) around RNAP (Naryshkin et al, 2000). To account for the full extent of the apparent DNA compaction in RPo at the PR promoter, it is necessary to invoke wrapping of upstream promoter DNA by nearly 300° around RNAP (Coulombe & Burton, 1999; Rivetti et al, 1999). Interactions between upstream promoter DNA and RNAP, presumably dependent on at least partial wrapping of upstream promoter DNA around RNAP, have been shown to affect the rate of formation of RPo (Davis et al, 2005; Ross & Gourse, 2005) and have been proposed to affect the rate of entry of DNA into, and unwinding of DNA in, the RNAP active centre cleft (Davis et al, 2005).
In this work, using AFM, we assess the effects of the promoter sequence, the RNAP -subunit carboxy-terminal domain (CTD) and the RNAP -subunit interdomain linker (-linker) on DNA compaction by RNAP. We find that DNA compaction depends on the sequence of the upstream region of the promoter, the presence of CTD and the presence and length of the -linker. Our results indicate that the sequence of the upstream region of the promoter affects DNA compaction not only through effects on CTD–DNA interaction but also through other effects—presumably effects on intrinsic DNA curvature. Our results further indicate that, in the absence of CTD–DNA interaction with upstream promoter DNA and intrinsic DNA curvature in the upstream region of the promoter, DNA compaction by RNAP is only 2–4 nm, consistent with the expectation from the modelled structure of RPo (Naryshkin et al, 2000). Overall, our results indicate that the massive, 30 nm, DNA compaction observed previously in RPo at the PR promoter (Rivetti et al, 1999) is not a general feature of RPo at all promoters.
Results
AFM measurements of DNA compaction in RPo
In this work, RNAP and RNAP derivatives were used to prepare RPo at the PR and lacUV5 promoters, or at substituted derivatives of these promoters (Fig 1A). Complexes were deposited onto freshly cleaved mica and imaged in air by AFM, and DNA contour lengths were measured (Fig 1B). Protein-induced apparent DNA compaction is defined as the difference between (i) the DNA contour length in the absence of protein (Fig 1C, top) and (ii) the DNA contour length of RNAP–DNA complexes (Fig 1C, bottom). This compaction was used as a quantitative indicator of protein-induced stable DNA wrapping, with a large compaction interpreted as a large protein-induced stable DNA wrapping (Fig 1D; Rivetti et al, 1999; Verhoeven et al, 2001; Heddle et al, 2004). Free contour length measurements were made by tracing the DNA path from end to end, as described previously (Rivetti & Codeluppi, 2001). The contour length of RNAP–DNA complexes was measured by tracing the DNA path from end to end and passing through the centre of the protein (Rivetti et al, 1999). In the case of protein–DNA complexes, the measurements are complicated because DNA in contact, or in close proximity, with the protein potentially can be hidden by tip-broadening and out-of-plane effects (Bustamante, 1993; Bustamante & Rivetti, 1996). This might result in underestimation of protein-free DNA contour lengths and overestimation of apparent DNA compaction. In the case of a large protein, such as RNAP, this effect can be up to 5 nm. Accordingly, in this work, an apparent DNA compaction is deemed significant as an indicator of stable DNA wrapping only if it is >5 nm.
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Figure 1
Atomic force microscopy measurements of DNA compaction. (A) Promoters analysed in this work. In each sequence, the transcription start site, the -10 and -35 hexamers are boxed. In PR-SUB(-463 to -36), heterologous DNA is shown in red, lower case; in lacUV5(UPprox), the consensus proximal UP-element subsite (Estrem et al, 1999) is shown in red; in lacUV5(UPfull), the consensus full UP element (Estrem et al, 1998) is shown in red. (B) Representative atomic force microscopy images of RPo formed with wild-type RNAP at the PR promoter (left) or the lacUV5(ICAP) promoter (right). Arrows point to individual complexes. The image scan size is 2  m. (C) Measurement of apparent DNA contour lengths in the absence (top) and presence (bottom) of RNAP. (D) Relationship between apparent DNA compaction and stable DNA wrapping. Grey dotted circle, RNAP; blue circle, tip broadening effect; black line, protein-free DNA; red dashed line, hidden DNA path; black dashed line, inferred DNA path through the centre of the RNAP. Apparent DNA compaction equals the difference in length between the red dashed line and the black dashed line. RNAP, RNA polymerase; RPo, RNA polymerase–promoter open complex.
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Surface interactions on deposition onto mica can also potentially cause an artefactual apparent DNA compaction (Rivetti et al, 1996). Accordingly, in this work, we used deposition conditions that allow complete configurational equilibration of DNA (Rivetti et al, 1996) as confirmed by the observation that mean-square end-to-end distances of DNA in the absence of RNAP were in the range predicted for an ideal worm-like chain polymer at equilibrium in two dimensions (data not shown; see Rivetti et al, 1996). It is thus believed that, under the conditions used, complexes were not significantly distorted by the deposition process (Bustamante & Rivetti, 1996).
DNA compaction depends on promoter sequence
Figure 2 and Table 1 present data for RNAP–promoter complexes at the PR and lacUV5(ICAP) promoters. For PR, an apparent DNA compaction of 30 0.4 nm was observed, consistent with previous results (Rivetti et al, 1999). By contrast, for lacUV5(ICAP), a strikingly smaller apparent DNA compaction, only 40.3 nm, was observed. Control experiments established that in the presence of nucleotides, 69% of the complexes formed at PR and 70% of the complexes formed at lacUV5(ICAP) can actively transcribe the downstream DNA. This indicates that most of the promoter-bound complexes were active RPo (supplementary Table S1 and Fig S1 online). We conclude that the extent of apparent DNA compaction in RPo is not constant from promoter to promoter, but, instead, depends on promoter sequence.
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Figure 2
The extent of DNA compaction depends on the sequence of the promoter. (A,B) Contour length distributions of DNA in the absence (hatched bars; top) and presence (grey bars; bottom) of RNAP are shown. Apparent DNA compaction is presented as means.e.m. RNAP, RNA polymerase.
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DNA compaction depends on upstream elements
To determine whether DNA compaction depends on sequence determinants in the upstream region of the promoter, we assessed DNA compaction in RNAP–promoter complexes at PR-SUB(-463 to -36), a PR derivative having a substitution replacing all sequences between positions -463 and -36 (Fig 1A). A large DNA compaction of 300.4 nm was observed for PR, whereas a strikingly smaller DNA compaction, of only 60.8 nm, was observed for PR-SUB(-463 to -36; Fig 3A; Table 1). Control experiments established that, in the presence of nucleotides, 77% of the complexes formed at PR-SUB(-463 to -36) were active RPo (supplementary Table S1 and Fig S1 online). We conclude that DNA compaction in RPo depends on sequence determinants in the upstream region of the promoter.
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Figure 3
The extent of DNA compaction depends on sequence determinants in the upstream region of the promoter and on CTD. Contour length distributions of DNA in the absence (hatched bars; top) and presence (grey bars; bottom) of RNAP, from experiments with promoter derivatives having substituted upstream regions (A–C) and with RNAP derivatives lacking both CTDs (D–F). Apparent DNA compaction is presented as means.e.m. CTD, -subunit carboxy-terminal domain; RNAP, RNA polymerase.
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Two classes of upstream region promoter elements have been defined: the UP-element subsite and the full UP element (Estrem et al, 1998, 1999; Gourse et al, 2000). The UP-element subsite (a short A/T-rich sequence centred in the -93, -83, -73, -63, -53 or, optimally, -43 region) stimulates transcription up to 200-fold through interactions with one RNAP CTD (Estrem et al, 1999; Gourse et al, 2000). The full UP element (two adjacent UP-element subsites, optimally one centred in the -53 region and the other centred in the -43 region) stimulates transcription up to 300-fold through interactions with two RNA CTDs (Estrem et al, 1998; Gourse et al, 2000).
To determine whether DNA compaction can be affected by a UP-element subsite or a full UP element, we assessed DNA compaction in RNAP–promoter complexes at the lacUV5(UPprox) and lacUV5(UPfull) promoters, respectively, a lacUV5 derivative containing a consensus UP-element subsite centred in the -43 region and a lacUV5 derivative containing a consensus full UP element centred in the -53/-43 region (Fig 1A; Estrem et al, 1998, 1999). A DNA compaction of only 20.5 nm was observed for the lacUV5(UPprox) promoter (Fig 3B; Table 1), whereas a DNA compaction of 210.7 nm was observed for lacUV5(UPfull) promoter (Fig 3C; Table 1). Control experiments established that, in the presence of nucleotides, 76% of the complexes formed at lacUV5(UPfull) were active RPo (supplementary Table S1 and Fig S1 online). We again conclude that DNA compaction in RPo depends strongly on sequence determinants in the upstream region of the promoter. We further conclude that a full UP element, but not a single UP-element subsite (at least not a single proximal UP-element subsite), represents a sequence determinant for large DNA compaction in RPo.
DNA compaction depends on the presence of the CTD
RNAP (subunit composition  'III  70) contains two identical -subunits, I and II. Each -subunit consists of (i) an amino-terminal domain (NTD) with determinants for dimerization and for interaction with ' and , (ii) an CTD with determinants for protein–DNA interaction with UP-element subsites and for protein–protein interactions with transcriptional regulators and (iii) a long, flexible interdomain linker (-linker; Blatter et al, 1994; Busby & Ebright, 1994). The long, flexible -linker allows CTD to occupy different positions relative to NTD—and thus relative to the remainder of RNAP—in different RNAP–promoter complexes (i.e. to interact nonspecifically with upstream DNA at a simple promoter, to interact with a UP-element subsite or UP element at a UP-element-subsite- or UP-element-containing promoter or to interact with an activator at an activator-dependent promoter; Busby & Ebright, 1994).
To determine whether DNA compaction is dependent on the presence of CTD, we assessed DNA compaction in RPo prepared using  CTDI/CTDII RNAP, an RNAP derivative lacking both CTDI and CTDII, at the PR and lacUV5(UPfull) promoters. At PR, a DNA compaction of 300.4 nm was observed with wild-type (WT)-RNAP, but a significantly smaller DNA compaction, of only 100.7 nm, was observed with CTDI/CTDII RNAP (Fig 3D; Table 1). Similarly, at lacUV5(UPfull), a DNA compaction of 210.7 nm was observed with WT-RNAP, but a significantly smaller DNA compaction, of only 130.6 nm, was observed with CTDI/CTDII RNAP (Fig 3E; Table 1). As expected, a DNA compaction of 40.8 nm was observed with CTDI/CTDII RNAP at lacUV5(ICAP) (Fig 3F; Table 1). We conclude that the extent of DNA compaction depends on the presence of CTD.
To determine whether DNA compaction is dependent on the presence of both CTDI and CTDII, we assessed DNA compaction in RPo assembled with CTDII RNAP, an RNAP derivative having CTDI but lacking CTDII (Estrem et al, 1999) at the PR and lacUV5(UPfull) promoters. At PR, only a small DNA compaction, of 130.4 nm, was observed with CTDII RNAP (a DNA compaction comparable with that observed with CTDI/CTDII RNAP; Fig 4A; Table 1). Similarly, at lacUV5(UPfull), only a small DNA compaction, of 130.5 nm, was observed with CTDII RNAP (Fig 4B; Table 1). As expected, a DNA compaction of 52.0 nm was observed with CTDII RNAP at lacUV5(ICAP) (Fig 4C; Table 1). We conclude that CTDI alone is insufficient to mediate large-scale DNA compaction.
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Figure 4
CTDI alone is insufficient to mediate large-scale DNA compaction. (A–C) Contour length distributions of DNA in the absence (hatched bars; top) and presence (grey bars; bottom) of RNAP, from experiments with CTDII RNAP derivatives. Apparent DNA compaction is presented as means.e.m. Because of the paucity and scatter of the data, the mean value in (C) represents the arithmetical average of the data. CTD, -subunit carboxy-terminal domain; RNAP, RNA polymerase.
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DNA compaction depends on the -linker
The -linker is 15 amino acids in length and comprises -residues from residue 236 to 251 (Blatter et al, 1994; Meng et al, 2000).
To determine whether DNA compaction is dependent on the presence and length of the -linker, we assessed DNA compaction in RPo assembled with 6-I/6-II RNAP, an RNAP derivative lacking six amino-acid residues of the -linker (-residues 235–241; Meng et al, 2000), and with 12-I/12-II RNAP, an RNAP derivative lacking 12 amino-acid residues of the -linker (-residues 235–247; Meng et al, 2000).
At PR, only a small DNA compaction, of 150.4 or 140.6 nm, was observed with 6-I/6-II RNAP or 12-I/12-II RNAP (a DNA compaction comparable with that observed with CTDI/CTDII RNAP; Fig 5A,B; Table 1). Similarly, at lacUV5(UPfull), only a small DNA compaction, of 130.6 or 121.1 nm, was observed with 6-I/6-II RNAP and 12-I/12-II RNAP (a DNA compaction comparable with that observed with CTDI/CTDII RNAP; Fig 5C,D; Table 1). We conclude that the -linker is required for large-scale DNA compaction.
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Figure 5
The extent of DNA compaction depends on the length of the -linker. (A–D) Contour length distributions of DNA in the absence (hatched bars; top) and presence (grey bars; bottom) of RNAP derivatives, from experiments with RNAP derivatives lacking residues of the -linker. Apparent DNA compaction is presented as means.e.m. -linker, -subunit interdomain linker; RNAP, RNA polymerase.
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The DNA bend angle is coupled to the DNA compaction
The DNA bend angle, defined as the deviation from linearity of the double helix, was measured for each complex by means of tangents drawn at the exit points of DNA from RNAP. The data are shown in supplementary Figs S3–S6 online. Overall, there is a good correlation between DNA compaction and bend angle. For example, the DNA bend angle of WT-RNAP at PR is 552.4°, which is consistent with an almost complete turn of the DNA around the RNAP at this promoter (see also figure 8 in Rivetti et al, 1999). Conversely, at lacUV5(ICAP), the measured DNA bend angle is 163.3°, which is consistent with the little DNA compaction observed at this promoter. At PR-SUB(-463 to -36) and lacUV5(UPprox) promoters, the DNA bend angles are 04.8° and 184.7°, respectively. These values correlate well with the little DNA compaction observed with these promoters. At lacUV5(UPfull), the measured DNA bend angle is 491.4°, which is again consistent with the 21 nm DNA compaction observed at this promoter. Thus, we conclude that a large DNA compaction associated with a high DNA bend angle is the result of DNA wrapping around the RNAP.
A statistically significant correlation between DNA compaction and DNA bend angle cannot be attributed for complexes assembled with CTD-RNAP mutants. This might be due to the intermediate DNA compaction observed in some of these cases, the presence of different types of complex as shown by the bimodal DNA contour length distributions and difficulties of obtaining narrow bend angle distributions.
Discussion
Our results establish that the extent of stable DNA wrapping in RPo depends on the sequence of the promoter and, in particular, on sequence determinants in the upstream region of the promoter (UP elements). The presence of CTD and an intact -linker is required to maintain extensive stable DNA wrapping. Our results further indicate that the sequence of the upstream region of the promoter can affect DNA wrapping even in the absence of CTD and thus even in the absence of CTD–DNA interactions. For example, RPo prepared using CTDI/CTDII RNAP shows an apparent DNA compaction of 130.6 nm at lacUV5(UPfull) but only 40.8 nm at lacUV5(ICAP) (Fig 3E,F). We infer that the sequence of the upstream region of the promoter can affect compaction not only through effects on CTD–DNA interaction but also through other effects. We suggest that these other effects involve intrinsic DNA curvature, noting that UP-element subsites and UP elements are A/T-rich sequences (Fig 1A; Ross et al, 1993) and that A/T-rich sequences are associated with intrinsic DNA curvature (Koo et al, 1986). In the absence of CTD–DNA interaction with upstream promoter DNA and of intrinsic DNA curvature in upstream DNA, stable DNA wrapping in RPo is small.
Overall, we show that tight, stable DNA wrapping is not a general feature of RPo, but rather depends on the promoter sequence. The tight, stable DNA wrapping observed at PR implies the presence, in the upstream region of this promoter, of sequence determinants that promote stable DNA wrapping—sequence determinants that, similar to the UP element in lacUV5(UPfull), make specific protein–DNA interactions with RNAP, favour intrinsic DNA curvature or both. A detailed analysis of the sequence determinants responsible for stable DNA wrapping at PR will be presented elsewhere.
Interestingly, as described in the supplementary information online, inactive promoter complexes (complexes in which RNAP is bound to the promoter but is unable to initiate RNA synthesis), in particular those formed at PR and lacUV5(UPfull), have a mean DNA compaction that is significantly smaller than that of the corresponding active RPo (supplementary Fig S2 and Table S2 online).
The results of photocrosslinking studies indicate that the CTDs can interact sequence-nonspecifically with the upstream region of the lacUV5 promoter, making sequence-nonspecific interactions with the DNA minor groove near positions -43, -53, -63, -73, -83 and -93 (Naryshkin et al, 2000). The results of promoter-truncation experiments indicate that the upstream region of the lacUV5 promoter can influence the association of RNAP (Ross & Gourse, 2005). How can this evidence for upstream interactions at lacUV5 be reconciled with the observed near-absence of apparent DNA compaction and thus the near-absence of stable DNA wrapping at lacUV5? We suggest that CTD interacts only transiently with the upstream region of lacUV5, making interactions sufficient to allow crosslinking and to allow effects on the association of RNAP, but not sufficient to yield measurable DNA compaction at equilibrium (as in imaging by AFM under the conditions used in this work; see the discussion in the first section of Results; see also Bustamante & Rivetti, 1996). In this regard, we note that (i) the photogenerated reactive species used in photocrosslinking studies is relatively long-lived, allowing efficient sampling of transient interactions (Naryshkin et al, 2000; N.N. & R.H.E., unpublished data), and (ii) rapid quenching of the photogenerated reactive species eliminates crosslinking of CTD with the upstream region of lacUV5 (N.N. & R.H.E., unpublished data). Thus, results from photocrosslinking expressly support the occurrence of transient, as opposed to stable, interactions of CTD with the upstream region of lacUV5.
An extended RNAP–DNA interaction can affect transcription in several, not mutually exclusive ways. A larger number of contacts between the RNAP and promoter DNA can increase the overall affinity of RNAP for the promoter (Aiyar et al, 1998; Estrem et al, 1998, 1999; Ross et al, 1993, 1998). An extended RNAP–DNA interaction, and corresponding DNA wrapping and wrapping-dependent distortion, also potentially can mechanically influence the isomerization from RNAP–promoter closed to RNAP–promoter open complex (Coulombe & Burton, 1999; Davis et al, 2005; Ross & Gourse, 2005). However, it should be emphasized that, as shown in this work, tight, stable DNA wrapping is not a general feature of all RPo at all promoters, but rather is strongly dependent on the promoter sequence.
Methods
DNA templates and proteins. The 1,054-bp-long DNA fragment carrying PR contains -DNA from –438 to +34 with respect to the PR start site, which is positioned 616 bp from the downstream end. The 963-bp-long DNA fragment PR-SUB(-463 to -36) contains -DNA from -35 to +34 with respect to the PR start site, which is positioned 500 bp from the downstream end. The 832-bp-long DNA fragment carrying lacUV5(ICAP) has the start site positioned 360 bp from the downstream end. The 1,191 and 1,050-bp-long DNA fragments carrying lacUV5(UPfull) have the start site positioned 706 and 602 bp from the downstream end, respectively. The 1,050-bp-long DNA fragment carrying lacUV5(UPprox) has the start site positioned 602 bp from the downstream end. Details about the preparation of DNA fragments and proteins are provided in the supplementary information online.
Complex formation and AFM imaging. RPo complexes were prepared by mixing 20 nM DNA with 20 nM RNAP in transcription buffer (20 mM Tris–HCl pH 7.9, 50 mM KCl, 5 mM MgCl2). The 10 l reaction was incubated at 37°C for 15 min. The reaction was diluted to 1–2 nM complexes in 20 l of deposition buffer (4 mM HEPES pH 7.4, 10 mM NaCl, 4 mM MgCl2) and deposited onto freshly cleaved mica. The sample was incubated for about 2 min before the surface was rinsed with water and dried with nitrogen. AFM imaging was carried out in air with the tapping mode using a Nanoscope III microscope (Veeco Digital Instruments, Santa Barbara, CA, USA).
Supplementary information is available at EMBO reports online (http://www.emboreports.org).
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Acknowledgements
We thank M. Thomas for plasmids and Centro Interfacoltà Misure for the AFM facility. This work was supported by the Italian Ministry of University and Scientific and Technological Research grant FIRB-RBNE01KMT9 to C.R. and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute investigatorship to R.H.E.
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