Skip to main content

Main menu

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
  • Submit
  • About
    • Editorial Board
    • PNAS Staff
    • FAQ
    • Accessibility Statement
    • Rights and Permissions
    • Site Map
  • Contact
  • Journal Club
  • Subscribe
    • Subscription Rates
    • Subscriptions FAQ
    • Open Access
    • Recommend PNAS to Your Librarian

User menu

  • Log in
  • My Cart

Search

  • Advanced search
Home
Home
  • Log in
  • My Cart

Advanced Search

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
Research Article

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Chin-Yu Chen, Chia-Chi Chang, Chi-Fu Yen, Michael T.-K. Chiu, and Wei-Hau Chang
  1. aInstitute of Chemistry and
  2. bGenomic Research Center, Academia Sinica, Taipei 115, Taiwan

See allHide authors and affiliations

PNAS January 6, 2009 106 (1) 127-132; https://doi.org/10.1073/pnas.0811689106
Chin-Yu Chen
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chia-Chi Chang
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chi-Fu Yen
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael T.-K. Chiu
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wei-Hau Chang
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: weihau@chem.sinica.edu.tw weihau40@gmail.com
  1. Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, November 18, 2008

  2. ↵1C.-Y.C., C.-C.C., and C.-F.Y. contributed equally to this work. (received for review March 26, 2008)

  • Article
  • Figures & SI
  • Info & Metrics
  • PDF
Loading

Abstract

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5′ end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5′ end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.

  • nanometry
  • structure
  • transcription
  • in-gel
  • single-molecule fluorescence

RNA polymerase II (pol II), a protein complex containing 12 subunits, Rpb1–Rpb12, of a total mass of ≈500 kDa and size ≈100–140 Å, is the enzyme machinery synthesizing mRNA in all eukaryotes (1). X-ray studies of pol II complexes (2–4) led to an atomic model containing structural elements with functional implications (Fig. 1A). In a transcribing pol II, between the “clamp” and “jaw” domain, lies a cleft (4) that harbors the active center, a straight duplex DNA and an RNA–DNA hybrid (position +1 to −8, +1 denoting the nucleotide addition site). The strand separation of RNA from DNA template occurs upstream of the hybrid at positions −9 and −10, facilitated by a set of protein loops including the “lid” domain as a driving wedge. Nascent RNA moves through an exit pore from the active center, crossing a saddle-like surface, beneath an “arch” bridging the clamp and wall (5).

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Pol II elongation complex. (A) Surface representation of a pol II elongation complex (PDB ID code 1Y1W) with structural elements highlighted: clamp in wheat, wall in violet, lid in green, rudder in yellow, fork loop 1 in orange, jaw in purple, and dock domain in aquamarine. Two putative RNA exit channels are indicated by red dash, labeled with 1 and 2. (B) The red star denotes a Cy5 on 10-nt RNA (GE2), situated next to the saddle. Green beads denote the Cy3 near the C terminus of Rpb4 in light pink or that of Rpb3 in cyan. The distance of Rpb4–GE2 is 82 Å and that of Rpb3–GE2 is 65 Å, respectively. (Inset) Cy3-CaM (orange) bound to CBP (light gray) extended from the C terminus of Rpb3 or Rpb4 in the presence of Ca2+ ions (yellow). (C) A design of triplet used for in-gel FRET. (Upper) Mixture of labeled and unlabeled pol II elongation complexes. Rpb3 or Rpb4 subunit is highlighted in pink; CaM is an orange dumbbell; Cy3 is green, and Cy5 is red. (Left) Pol II elongation complex with RNA unlabeled, a mixture of Cy3-CaM bound or unbound in the upper band, free Cy3-CaM in the lower band. (Middle) Pol II elongation complexes labeled with Cy5-RNA and Cy3-CaM. (Right) Same as Middle except unlabeled CaM is used. (D) Immobilized single molecules of pol II elongation complexes on a coated slide. (Left) Donor only, labeled with Cy3-CaM. (Middle) Donor and acceptor, with Cy3-CaM and Cy5-RNA. (Right) Acceptor only, with Cy5-RNA. A and B were prepared by PyMOL (50) (www.pymol.org).

How does pol II instruct the nascent RNA to exit beyond the saddle? Is there a unique path on pol II connecting the active center to its exterior that nascent RNA may follow? To date, insights into the RNA exit have come from analysis of pol II surface charge distribution: two positively charged grooves, on either side of the “dock domain” (Fig. 1A), can accommodate ssRNA (5). One groove, putatively referred as “exit channel 1,” runs around the base of the clamp, leading toward the stalk of subcomplex Rpb4–Rpb7, which can bind RNA via its ribonucleoprotein fold (6, 7). The other groove, termed “exit channel 2,” runs down the back side of pol II, through Rpb3 and Rpb11, leading toward Rpb8, a subunit equally competent in RNA binding by its single-strand nucleic acid-binding motif. Intriguingly, exit channel 1 would cause the RNA to bend sharply, implying that channel 2 is energetically favored for RNA binding. Yet, evidence in support of the channel 1 hypothesis has come from observations of the nascent RNA cross-linking to Rpb7 subunit of pol II (8).

The channel 1 hypothesis is tantalizing, given that the Rpb4–Rpb7 subcomplex is dispensable for RNA synthesis in yeast (9). Attempts to identify the exit channel by X-ray studies of the pol II elongation complex failed to detect RNA longer than 10 nt (10). An alternative approach is thus required to address this issue. Fluorescence resonance energy transfer (FRET) is a spectral ruler (11) to gauge distance between 1 and 10 nm (12), ideal for mapping pairs of probes on a complex (13, 14) as large as pol II. Indeed, FRET analysis of Escherichia coli RNA polymerase, a counterpart of pol II in bacteria, pioneered by Ebright and coworkers (15–18), revealed the spatial organization of the promoter complex, retention of σ70 and a DNA-scrunching mechanism at initiation. Those FRET studies of E. coli RNA polymerase were facilitated by assembling the enzyme complex from its individual subunits, which could be specifically dye-labeled before reconstitution.

A similar FRET approach to pol II has been impeded by lack of a reconstituting system, except that the dissociation of Rpb4–Rpb7 from core pol II can be exploited (9). Here, we introduce a simple scheme for specifically labeling virtually any subunit in a TAP-tagged (tandem affinity purification) protein complex (19, 20). Briefly, Cy3-conjugated calmodulin (CaM) is used to poise a Cy3 dye near the C terminus of a TAP-tagged pol II subunit by its binding to the CaM-binding peptide (CBP) on the subunit (Fig. 1B). With a Cy5 dye attached to the 5′ end of RNA, our scheme allows us to test the hypothesis about the RNA exit channel on pol II by FRET analysis. If channel 1 is preferred, we would expect an increase in FRET efficiency between Cy3 near the C terminus of Rpb4 and Cy5 on the 5′ end of RNA, as the RNA extends. Conversely, should channel 2 be preferred, there would be an increase in FRET efficiency between Cy3 near the C terminus of Rpb3 and Cy5 on the 5′ end of RNA with extension of the RNA. In the present work, two independent FRET measurements are performed: “in-gel FRET” (Fig. 1C) (13, 14) and “single-molecule FRET” (Fig. 1D) (21, 22). The former is a bulk measurement, facilitated by separation of pol II from unbound Cy3-CaM or Cy5-RNA in a native gel, whereas the latter allows real-time recording of the “double-labeled” complex to reveal dynamics and distributions. For simplicity, we employ the following notation to denote the sites of labeling and the corresponding FRET efficiency measurement in the subsequent text. For instance, “FRET efficiency between Cy3 near the C terminus of Rpb4 subunit of pol II and Cy5 attached at the 5′ end of GE2 RNA (10 nt)” is referred to as “FRET of Rpb4–GE2.”

Results

Activity of Labeled Pol II Elongation Complexes.

In this work, pol II elongation complexes are obtained by assembling 12-subunit pol II [supporting information (SI) Fig. S1A] with nucleic acid scaffold, to mimic complexes at discrete points along the trajectory of elongation. The hybridizing region of RNA with the template DNA is kept to 8 bp (10), whereas the 5′ nonhybrid growing end is 2, 9, 18, termed GE2, GE9, GE18, respectively (GE denotes “growing end”). RNA, together with template DNA, forms a stable complex with pol II (5, 10). The maximum number of nucleotides in the upstream nonhybridizing RNA was chosen such that it may fully span the channel. [Note that each candidate channel measures ≈45 Å from the saddle to its outlet on core pol II (10 subunits), and hence both channels can accommodate 13–16 nt (5).] An in vitro transcription assay was conducted to assure that pol II elongation complexes retain their ability to extend RNA, either with Cy5 attached to the 5′ end of RNA (Fig. S1B) or with a few millimeter calcium ions (Fig. S1C). It was important to ensure that calcium ions, required for CaM–CBP interactions, did not interfere with pol II elongation because such ions abolish transcription activity in a related system (23). Based on the unaltered function of the pol II elongation complexes, we believe that the labeling used in our experiments would not cause significant structural perturbations of the complexes.

In-Gel FRET Analysis.

Bulk FRET measurements in solution have met practical obstacles. The sensitivity of fluorescence spectrometers is typically in the concentration range of ≈micromolar, implying that ≈milligrams of fluorescence-labeled pol II are required just to generate a data point. We therefore resorted to an alternative bulk method by using a native gel, requiring less material. In native gel, later referred to as in-gel, pol II complexes can be separated from the unbound Cy3-CaM (≈20 kDa), in the low-molecular mass region (Fig. 2A and C). The upper band corresponds to a mixture of labeled and unlabeled pol II complexes. [This upper band appeared as a singlet or a doublet, depending on the phosphorylation state of the CTD of the Rpb1 in pol II (24–26)]. fBand fOA were measured and summarized (see Gel Quantitation in SI Materials and Methods and Tables S1 and S2).

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

In-gel FRET efficiencies as a function of the length of RNA. (A) Gel image of triplets scanned in the Cy3 channel. (Upper) From Cy3-CaM on Rpb3 subunit in pol II elongation complex. (Lower) From unbound Cy3-CaM. Lanes 1–3, 4–6, and 7–9 represent three triplets of elongation complexes containing GE2 (10 nt), GE9 (17 nt), and GE18 (26 nt), respectively. (B) X–Y plot of FRET efficiencies between Cy3 on Rpb3 and Cy5 on RNA, extracted from replica images of A, as a function of the length of RNA. ■, Rpb3_1: Cy3-CaM labeling on pol II is 10% and RNA binding 40% (fB). ●, Rpb3_2: Cy3 labeling is 30% and RNA binding 40% (fB). (C) Same as in A except Cy3-CaM is on Rpb4. (D) Extracted from replica of C. ■, Rpb4_1: Cy3 labeling was 5% and RNA binding 55% (fB). ●, Rpb4_2: Cy3 labeling was 12% and RNA binding 40% (fB).

In-gel FRET efficiencies between a pol II subunit and RNA of various lengths, GE2, GE9, and GE18, were compared. Raw FRET efficiencies were measured as described in Materials and Methods (also in SI Materials and Methods). As the RNA extended from GE2 to GE18, an increase in raw FRET between Rpb4 was observed repeatedly (Fig. 2D), immediately suggesting that nascent RNA is leading toward the Rpb4–Rpb7. By contrast, no gain in raw FRET was observed between Rpb3 and RNA as the length of the RNA was extended (Fig. 2B). Average authentic in-gel FRET efficiencies were calculated by using measured fBand fOA and converted into distances (Table 1). To challenge whether those distances were plausible, in-gel FRET efficiencies between Cy3-DNA and Cy5-RNA of various lengths were measured (Fig. S2) and found to be consistent with the triangulation results based on Rpb3–RNA and Rpb4–RNA distances (Table 1 and Table S2).

View this table:
  • View inline
  • View popup
Table 1.

Results from average in-gel FRET and single-molecule FRET (smFRET) measurements

Single-Molecule FRET Analysis.

Even though in-gel FRET provided reliable distances for tracking nascent RNA in the pol II elongation complex, replica FRET experiments were performed with the single-molecule method for cross-validation. In the immobilized single-molecule scheme, dual-labeled complexes are selected; thus, the information of dye-labeling efficiency, a bulk quantity, is dispensable. Single-molecule time traces of raw fluorescence intensities and calculated FRET efficiencies are shown in Fig. S3, and constructed histograms are in Fig. 3.

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Single-molecule FRET histograms. (A) Reconstructed from many leakage-QE-corrected time traces of FRET efficiencies, between Cy3-Rpb3 and Cy5 attached to the 5′ end of RNA of various lengths: GE2 (10 nt), GE9 (17 nt), and GE18 (26 nt). (B) Same as A except Cy3-CaM is on Rpb4.

For Rpb3–GE2, the FRET histogram can be fitted to two Gaussian distributions, centering at 0.49 (Fig. 3A P5) and 0.40 (Fig. 3A P4), respectively. As the RNA extends to GE9, the FRET histogram shifts toward the low-FRET regime with the major distribution centering at 0.32 (Fig. 3A P2), indicating that the distance between Rpb3 and GE9 is longer than that between Rpb3 and GE2. For Rpb4–GE2, the FRET histogram shows a major distribution centering at 0.17 (Fig. 3B P1). As the RNA extends to GE9, the FRET histogram shifts toward the high-FRET regime, and it can be fitted to two Gaussian distributions with the major one centering at 0.3 (Fig. 3B P3), indicating that Rpb4 is closer to GE9 than to GE2. As the RNA extends further to GE18, changes of FRET values follow the same trend, while broadening in the distributions is observed, and minor populations of “anomalous” FRET emerge: high for Rpb3 (Fig. 3A P6) and low for Rpb4 (Fig. 3B P4). The peak FRET values of the major single-molecule populations are summarized (Table 1), in good agreement with the corresponding in-gel FRET efficiencies that virtually indistinguishable distances, within 5-Å errors, can either be generated from single-molecule data or from in-gel data (Table 1). Thus, single-molecule FRET data also support that the majority of nascent RNA molecules, if not all, exit through channel 1 on pol II.

Structural Mapping of RNA Exit Based on Single-Molecule FRET RNA GE2 (10 Nucleotides).

By using single-molecule FRET efficiencies, 0.49 for Rpb3–GE2 (Fig. 3A P5) and 0.17 for Rpb4–GE2 (Fig. 3B P1) and a Förster distance R0 ≈60 Å for Cy3–Cy5 (22, 27–29), distances of 61 Å (1.01 R0) and 78 Å (1.30 R0) are obtained for Rpb3–GE2 and Rpb4–GE2, respectively (Table 1), agreeing well with 64 Å and 82 Å, the corresponding distances in the crystal structure (10). An additional single-molecule FRET between Cy5–GE2 and Cy3–DNA, termed DNA-GE2, is measured, and a shorter Förster distance R0 ≈48 Å for Cy3–Cy5 is required to fit with the corresponding distance (≈50 Å) in the crystal structure (Fig. S4). Interestingly, such a reduction of Förster distance has also been observed in in-gel data of DNA-GE2 (Table 1).

Localization of the 5′ End of the RNA GE9 (17 Nucleotides).

Assuming that Förster distance R0 ≈60 Å is applicable, distances of 68 Å and 69 Å are obtained for Rpb3–GE9 and Rpb4–GE9, respectively, and are summarized in Table 1. By allowing a ±5 Å error, a unique site defined by a set of amino acids residing in the presumed exit channel 1 on core pol II is found by triangulation (Table S3, and salmon sphere in Fig. 4). The position of GE9, predicted based on the triangulation of Rpb3–GE9 and Rpb4–GE9, spans a distance of 60 ± 5 Å to the Cy3 site of DNA (Cy3 dye attached between G11 and T12), consistent with the distance calculated from the single-molecule FRET data of DNA-GE9 with Förster distance R0 ≈48 Å (Table 1 and Fig. S4). The distance from the 5′ end of the GE2 (10 nt) to that of GE9 (17 nt) is determined to be 25–30 Å. As expected, this span is capable of accommodating 7–11 nt. Henceforth, we refer to exit channel 1 as the exit channel.

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Locations of the 5′ end of RNA. The 5′ end of GE9 (17 nt) is in salmon, next to the dock domain in aquamarine; the 5′ end of GE18 (26 nt) is in orange, on the Rpb7 ribonucleoprotein-binding domain in pink; also the 5′ end of GE2 (10 nt) is in red, the C terminus of Rpb3 in cyan, and that of Rpb4 in violet. The figure was generated by PyMOL (50) (www.pymol.org) and the program O (51).

GE18 (26 Nucleotides) and RNA Dynamics.

By using single-molecule FRET values, 0.18 for Rpb3–GE18 (Fig. 3A P1) and 0.62 for Rpb4–GE18 (Fig. 3B P5), distances of 77 Å and 55Å are obtained, respectively (Table 1). Triangulation with these distances identifies a site on the ribonucleoprotein-binding domain of Rpb7 (Table S3) (6, 10), shown as an orange sphere (Fig. 4). The distance between the 5′ end of GE18 (26 nt) and Cy3 site of DNA (Cy3 dye attached between G11 and T12) is predicted to be 65 ± 5 Å, resulting in low FRET efficiencies, challenging to be detected by our single-molecule instrument (Table 1). The finding that GE18 (26 nt) contacts Rpb7 lines up with the previous study of the 5′ end of nascent RNA of 23–29 nt cross-linking to Rpb7 (8). The trajectory from the exit pore to the Rpb7 site deviates slightly from that of the exit channel, which would produce an energy penalty that could be compensated by RNA interacting with the ribonucleoprotein-binding domain. Interestingly, as the RNA extends to GE18 (26 nt), the distribution in the FRET histogram exhibits a broadening (Fig. 3 A and B), regardless on which subunit Cy3 is placed. Such broadening, originating from fluctuations in the time traces (Fig. S3 C and G), can be a signature of RNA flexibility because of its dislodging from pol II.

Discussion

Principal Findings.

The RNA exit channel has been a hypothetical entity in pol II elongation complex structure, on which the existence of two charged grooves leads to different models of the RNA exit pathway. In this work, by introducing a simple method to label the pol II subunit, we test the models by measuring the FRET efficiencies between a donor on a subunit and an acceptor on the 5′ end of RNA. Observations of markedly different trends of change of FRET efficiencies vs. RNA length in a native gel prove that the RNA exit channel leads toward Rpb4–Rpb7, not Rpb3–Rpb11. Quantitative FRET analysis shows that in-gel data are both self-consistent and in agreement with single-molecule data. By identifying amino acids in the vicinity of the 5′ end of RNA (Table S3), using distances calculated from single-molecule data and triangulation, we map the track of nascent RNA on pol II, which bends ≈90° from the direction of the DNA template. Such a bending presents a remarkable structure feature that can prevent nascent RNA from meeting with the DNA template. The location of the 17-nt RNA enables us to predict which nucleotide would be the last before nascent RNA extrudes from the body of core pol II. The distance between the 17-nt RNA to the outlet of channel is measured to be ≈15–20 Å, capable of accommodating 4–6 nt (5, 10), suggesting that the last nucleotide must lie between 21 and 23 nt, and RNA longer than 23 nt must extrude into the exterior of core pol II. Such a picture is remarkably consistent with a previous observation that the 5′ end of nascent RNA could cross-link to Rpb1 when RNA was shorter than 21 nt (8). As RNA extends to 26 nt, its 5′ end could contact a site within the ribonucleoprotein-binding domain in Rpb7 (Fig. 4), which also confirms the cross-linking study (8).

Remarks on FRET-Based Structural Biology.

Our study represents a case where a judicious choice of sites for a FRET pair is crucial for generating interpretable FRET data for “molecular nanometry,” to complement the high-resolution study of protein complexes. In the literature, single-molecule FRET has been most restricted to studies on revealing molecular heterogeneity and/or dynamics. With this regard, our work serves as a milestone in the application of single-molecule FRET to structural biology of protein complexes. In this study, the awesome power of the C terminus labeling scheme has not been fully harnessed. In principle, generation of a dozen FRET distances to a site on pol II, because there are a dozen of subunits in pol II, can help solve the structure problem in an overdeterministic fashion. Although both in-gel FRET and single-molecule FRET provide equally good information in our case, the single-molecule approach is preferred for practical reasons. First, the single-molecule method requires much less material, so it may be applicable to scarce eukaryotic complexes. Second, the conversion of raw in-gel FRET data to distances is laborious because it requires accurate characterization of binding efficiencies and optical properties of the reagents (Table S2). Nevertheless, one caution about our single-molecule experiments is Cy3-CaM falling off from pol II because the measurements were carried out in a picomolar concentration whereas the affinity between CaM and CBP is ≈nanomolar.

Biological Significance.

In the RNA exit channel, the interactions between nascent RNA and pol II in the region between 10 and 17 nt are expected to be very strong (30), for it is known that such interactions contribute to the stability of an elongation complex in prokaryotic RNA polymerase. Recent structural studies on the complexes formed by TFIIB with pol II have shown that the N-terminal segment of TFIIB, termed the “B finger,” reaches into the catalytic center of pol II exactly through a groove (31–34), once presumed to be the RNA exit channel and proved to be true in this study. It is thus evident that the B finger of TFIIB will run into the advancing RNA. If the TFIIB overcomes the RNA, the initiation will be aborted; if the RNA continues to advance, pol II will escape from the promoter. By comparing this scenario with that in E. coli transcription, a conserved strategy is found in E. coli: the nascent RNA pushes away a protein linker between domains 3 and 4 of the σ factor that preoccupies the RNA exit channel on RNA polymerase so that transit from initiation to elongation may occur (35, 36). As the RNA extends to 26 nt, it becomes more flexible yet continues to reach out for Rpb7. Why would RNA take the route via the subcomplex Rpb4–Rpb7? Perhaps Rpb4–Rpb7 serves as a scaffold to arrange a meeting between the nascent mRNA of ≈30 nt and its 5′ end-capping machinery (37). The latter is known to be recruited by the phosphorylated form of the CTD of Rpb1 subunit, a domain residing underneath Rpb4–Rpb7, so that transcription and mRNA processing can be efficiently coupled.

Reconciliation.

While our article was in revision, an independent single-molecule FRET study of a pol II elongation complex was published by Michaelis and coworkers (38), who labeled the dissociable subcomplex Rpb4–Rpb7 and reconstitution pol II of 12 subunits. Both studies support that the nascent RNA exits from pol II through channel 1. However, there seems to be a discrepancy as to where 26-nt RNA could contact. Contrary to our observation that the 5′ end of 26-nt RNA contacts Rpb7 in most elongation complexes, Michaelis and coworkers have suggested that the 5′ end of 26-nt RNA can occupy the “dock domain” on pol II (38). Intriguingly, our single-molecule data of 26-nt RNA reveal minor populations of anomalous FRET for Rpb3–GE18 (Fig. 3A P6) and Rpb4–GE18 (Fig. 3B P4) as well. Triangulation based on these anomalous distances predicts that the 5′ end of 26-nt RNA can reside on the dock domain, as suggested by the Michaelis group. By Boltzmann statistics, we estimate that there is an energy gap, ≈1–2 kBT per complex, between the Rpb7-contacting complex and the dock domain-contacting complex. Such a gap could be partially addressed by the bending of nucleic acids. As shown in the model of RNA exit (Fig. 4), RNA bent toward the dock domain-contacting position requires larger angles than that bent toward Rpb7 and thus is energetically unfavorable because the physics of bending RNA demands energy of kBT(Δθ)2(ξ/2L), where ξ is the persistent length of ssRNA, ≈1–1.4 nm, and L is the contour length of the ssRNA (39, 40). Of course, interactions of RNA–Rpb7 and those between RNA and dock domain must be taken into account to complete the analysis. We speculate that systematic variations in experimental conditions may influence the behavior of RNA in the pol II elongation complex. For instance, certain cations, ammonium, zinc, magnesium, and calcium, are either present or absent in the two studies. Some of these ions are known to play roles in fine-tuning the conformation of nucleic acids and/or proteins. Indeed, some can alter the elongation activity of pol II (41), and some can modulate the folding capacity of nucleic acids (42). The subtle effects of ions in the context of transcription merit further investigation, best pursued by single-molecule experiments to reveal energy landscape.

Materials and Methods

Construction of Yeast Strains and Protein Purification.

RNA polymerase II.

TAP-tagged yeast strains (Saccharomyces cerevisiae) were generated according to standard procedures (6, 43). Yeast cells expressing TAP-tagged Rpb3 or Rpb4 were grown and fractionated as described (6, 43) except a washing step with high concentration of potassium chloride (6, 44) was recruited to deplete TFIIF from pol II before the elution by Tobacco Etch Virus enzyme cleavage.

CaM.

To make a dye-CaM, a human CaM II (hCaM) was cloned into a pET22b vector (Novagen), in which the C terminus was His6-tagged, and the 3rd amino acid, aspartic acid, was mutated to a cysteine (45, 46) for maleimide-Cy3 conjugation. The cysteine mutant of the hCaM was overexpressed in E. coli. strain BL21 (DE3), purified with a Ni–nitrilotriacetic acid affinity column (Qiagen), and labeled with maleimide-Cy3 according to a standard protocol (GE Healthcare).

In-gel FRET Measurements and Data Reduction.

Pol II labeled with Cy3-CaM was separated from free Cy3-CaM based on molecular mass in a native gel made with gradient polyacrylamide (4–20%; Invitrogen). Likewise, DNA or RNA bound to pol II was separated from free DNA or RNA, in the low-molecular mass band. Electrophoresis was performed in TBE buffer containing 3 mM Ca2+ at 120 V for 1.5 h. The wet gels were immediately scanned in a Typhoon 9400 scanner (GE Healthcare) equipped with a 532-nm laser for Cy3 excitation and a 580-nm emission filter to collect Cy3 fluorescence. The fluorescence intensities of the complex bands were integrated, and background was subtracted by using ImageQuant TL software. The accuracy of pipetting and loading among three lanes in the “triplet” (see SI Materials and Methods) was critical and monitored by the fluorescence intensities of the unbound Cy3-CaM band in the low-molecular mass region: any gel containing a triplet in which the free Cy3-CaM in the left (donor only) and that in the middle lane (donor and acceptor) differed >4% was discarded. The efficiency of energy transfer, E, was determined from the extent of Cy3 quenched by Cy5 in the double-labeled complexes compared with donor only complexes. We determined the “raw” energy transfer efficiency ER in-gel (47) according to Eq. 1, Embedded Image where ID and IDA were the intensities of the donor-only (no energy transfer) and the donor and acceptor complexes, respectively. ER, the raw energy transfer efficiency, was converted to EA, the “authentic” energy transfer efficiency, according to Eq. 2, Embedded Image where f is the effective acceptor-labeling efficiency (see Table S3). fB, the fraction of pol II that contains RNA, namely binding efficiency of RNA to pol II, was determined according to Cy5 intensities in the upper and the lower bands according to Eq. 3, Embedded Image where ICy5u is the Cy5 signal appearing in the upper band, and ICy5l is the Cy5 signal appearing in the lower band. The effective acceptor labeling efficiency f could be obtained according to Eq. 4, Embedded Image where fOA was the optically active fraction of RNA.

Single-Molecule FRET Measurements.

A total internal reflection fluorescence microscope was built for single-molecule imaging (SI Materials and Methods) (46, 48) and quantum efficiency-calibrated (Fig. S5). Preformed elongation complexes consisting of Cy5-RNA, incubated with 5-fold excess of Cy3-CaM and diluted to 10 pM in transcription reaction buffer containing an imaging mixture, were immobilized on the functionalized slide surface through the biotinylated DNA template. The imaging mixture contained 10 mM Tris acetate (pH 7.5), 0.4% glucose, 2 mM Trolox (Fluka), 0.1 g/mL glucose oxidase (Sigma), and 0.02 mg/mL catalase, to remove solution oxygen and reduce blinking (49). Data were obtained with an alternative excitation sequence: 633 nm on (5 s) off, 532 nm on (400 s) off, with 0.3- to 0.5-s exposure per frame. Measurements were performed at 25 °C. Candidate molecules that were Cy3–Cy5 dual-labeled were selected based on Cy3 spot centroid colocalizing with Cy5 found through preexcitation in the first 10 frames. The simultaneous time trajectories were extracted from the same set of centroids and screened by checking for concomitant increase in Cy3 signal with Cy5 decrease or concomitant Cy5 signal decrease with Cy3 photobleaching. The jiggling of the spot positions between frames was fixed by extracting intensities from new centroids in the vicinity of the centroids in the previous frame. Donor and acceptor signals were converted to FRET efficiency according to Eq. 5, where IA and ID denoted the background-subtracted fluorescence intensities of the acceptor and the donor, respectively (β is the leakage of Cy3 signal into Cy5 channel; γ is the ratio of quantum efficiencies of the two channels). Distances were calculated from FRET efficiencies according to Eq. 6. Data collection was conducted with Andor software, and subsequent processing was conducted with customer-written IDL programs (IDL 6.3; ITT). Embedded Image Embedded Image Various pol II elongation complexes were examined: Rpb3–GE2, Rpb3–GE9, Rpb3–GE18, Rpb4–GE2, Rpb4–GE9, Rpb4–GE18. From ≈50 to 100 molecules, a histogram of corrected FRET efficiencies was constructed by including all data points of each the time trace of each molecule until Cy5 was bleached or Cy3-Cy5 codisappeared, and fitted with multiple Gaussian distributions.

Search of the Site in the Crystal Structure by Triangulation.

Averaged authentic in-gel FRET or single-molecule FRET efficiencies at the peaks in the major distribution were used to calculate distances. The distance of Rpb3–GE2 (Rpb4–GE2) in pol II elongation complex crystal structure [Protein Data Bank (PDB) ID code 1Y1W] was used to determine the Förster distance, by which the distances of Rpb3–GE9 (Rpb4–GE9) and Rpb3–GE18 (Rpb3–GE18) were derived. Triangulation was performed by searching atoms in the PDB (1Y1W) (10) that satisfied the distances with a given error. Such a “closure-error triangulation” scheme selected atoms within the intersection of two shells: vertex 1, distance from vertex 1, errors in distance; and vertex 2, distance from vertex 2, errors in distance. In the present work, vertex 1 was the last atom in the C terminus of Rpb3, and vertex 2 that of Rpb4.

Acknowledgments

We thank M.-J. Wang and Dr. Joan Chen [Institute of Biomedical Sciences, Academia Sinica (AS)] for providing the human CaM II clone; Dr. Yu-Ju Chen (Institute of Chemistry, AS), for mass spectroscopy; Tommy Setiawan and Y.-P. Weng in the Chang laboratory for CaM expression and purification. We are also grateful for critical discussions with Dr. Sunney Chan (AS and Caltech), Dr. David Bushnell (Stanford), and Prof. Averell Gnatt (University of Maryland, Baltimore). Dr. Chin-Yu Chen has been supported by a National Science Council postdoctoral fellowship and is currently supported by an AS postdoctoral fellowship. This work was supported by AS Grants AS95IC1 and AS-95-TPB06 and National Science Council of Taiwan Grants NSC94-2113-M-001-015, NSC95-2113-M-001-031, and NSC94-2627-B-001-003 (all to W.-H.C.).

Footnotes

  • 2To whom correspondence should be addressed at:
    Institute of Chemistry, Academia Sinica: 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan.
    E-mail: weihau{at}chem.sinica.edu.tw or weihau40{at}gmail.com
  • Author contributions: W.-H.C. designed research; C.-Y.C., C.-C.C., and C.-F.Y. performed research; C.-Y.C., C.-C.C., C.-F.Y., M.T.-K.C., and W.-H.C. analyzed data; and C.-Y.C. and W.-H.C. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0811689106/DCSupplemental.

  • Freely available online through the PNAS open access option.

  • © 2008 by The National Academy of Sciences of the USA

References

  1. ↵
    1. Kornberg RD
    (2007) The molecular basis of eukaryotic transcription. Proc Natl Acad Sci USA 104:12955–12961.
    OpenUrlFREE Full Text
  2. ↵
    1. Cramer P,
    2. et al.
    (2000) Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288:640–649.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Cramer P,
    2. Bushnell DA,
    3. Kornberg RD
    (2001) Structural basis of transcription: RNA polymerase II at 2.8 Å resolution. Science 292:1863–1876.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Gnatt AL,
    2. Cramer P,
    3. Fu J,
    4. Bushnell DA,
    5. Kornberg RD
    (2001) Structural basis of transcription: An RNA polymerase II elongation complex at 3.3 Å resolution. Science 292:1876–1882.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Westover KD,
    2. Bushnell DA,
    3. Kornberg RD
    (2004) Structural basis of transcription: Separation of RNA from DNA by RNA polymerase II. Science 303:1014–1016.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bushnell DA,
    2. Kornberg RD
    (2003) Complete, 12-subunit RNA polymerase II at 4.1- Å resolution: Implications for the initiation of transcription. Proc Natl Acad Sci USA 100:6969–6973.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Craighead JL,
    2. Chang WH,
    3. Asturias FJ
    (2002) Structure of yeast RNA polymerase II in solution: Implications for enzyme regulation and interaction with promoter DNA. Structure 10:1117–1125.
    OpenUrlPubMed
  8. ↵
    1. Ujvari A,
    2. Luse DS
    (2006) RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit. Nat Struct Mol Biol 13:49–54.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Edwards AM,
    2. Kane CM,
    3. Young RA,
    4. Kornberg RD
    (1991) Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J Biol Chem 266:71–75.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Kettenberger H,
    2. Armache KJ,
    3. Cramer P
    (2004) Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol Cell 16:955–965.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Stryer L,
    2. Haugland RP
    (1967) Energy transfer: A spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726.
    OpenUrlFREE Full Text
  12. ↵
    1. Hillisch A,
    2. Lorenz M,
    3. Diekmann S
    (2001) Recent advances in FRET: Distance determination in protein–DNA complexes. Curr Opin Struct Biol 11:201–207.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Kerppola TK
    (2001) The bright future of fluorescence. Methods 25:1–3.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Ramirez-Carrozzi V,
    2. Kerppola T
    (2001) Gel-based fluorescence resonance energy transfer (gelFRET) analysis of nucleoprotein complex architecture. Methods 25:31–43.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kapanidis AN,
    2. et al.
    (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314:1144–1147.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Margeat E,
    2. et al.
    (2006) Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. Biophys J 90:1419–1431.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Kapanidis AN,
    2. et al.
    (2005) Retention of transcription initiation factor σ70 in transcription elongation: Single-molecule analysis. Mol Cell 20:347–356.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Naryshkin N,
    2. Revyakin A,
    3. Kim Y,
    4. Mekler V,
    5. Ebright RH
    (2000) Structural organization of the RNA polymerase–promoter open complex. Cell 101:601–611.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Rigaut G,
    2. et al.
    (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Puig O,
    2. et al.
    (2001) The tandem affinity purification (TAP) method: A general procedure of protein complex purification. Methods 24:218–229.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Weiss S
    (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat Struct Biol 7:724–729.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Ha T
    (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Lue NF,
    2. et al.
    (2005) Telomerase can act as a template- and RNA-independent terminal transferase. Proc Natl Acad Sci USA 102:9778–9783.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Li Y,
    2. Kornberg RD
    (1994) Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II. Proc Natl Acad Sci USA 91:2362–2366.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Gileadi O,
    2. Feaver WJ,
    3. Kornberg RD
    (1992) Cloning of a subunit of yeast RNA polymerase II transcription factor b and CTD kinase. Science 257:1389–1392.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Feaver WJ,
    2. Gileadi O,
    3. Li Y,
    4. Kornberg RD
    (1991) CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell 67:1223–1230.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Norman DG,
    2. Grainger RJ,
    3. Uhrin D,
    4. Lilley DM
    (2000) Location of cyanine-3 on double-stranded DNA: Importance for fluorescence resonance energy transfer studies. Biochemistry 39:6317–6324.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Iqbal A,
    2. et al.
    (2008) Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids. Proc Natl Acad Sci USA 105:11176–11181.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Hohng S,
    2. Joo C,
    3. Ha T
    (2004) Single-molecule three-color FRET. Biophys J 87:1328–1337.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Nudler E,
    2. Gusarov I,
    3. Avetissova E,
    4. Kozlov M,
    5. Goldfarb A
    (1998) Spatial organization of transcription elongation complex in Escherichia coli. Science 281:424–428.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Bushnell DA,
    2. Westover KD,
    3. Davis RE,
    4. Kornberg RD
    (2004) Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Å. Science 303:983–988.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Chen HT,
    2. Hahn S
    (2004) Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: A model for the structure of the PIC. Cell 119:169–180.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Chen HT,
    2. Hahn S
    (2003) Binding of TFIIB to RNA polymerase II: Mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. Mol Cell 12:437–447.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Chen HT,
    2. Warfield L,
    3. Hahn S
    (2007) The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nat Struct Mol Biol 14:696–703.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Murakami KS,
    2. Masuda S,
    3. Campbell EA,
    4. Muzzin O,
    5. Darst SA
    (2002) Structural basis of transcription initiation: An RNA polymerase holoenzyme–DNA complex. Science 296:1285–1290.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Vassylyev DG,
    2. Vassylyeva MN,
    3. Perederina A,
    4. Tahirov TH,
    5. Artsimovitch I
    (2007) Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448:157–162.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Proudfoot NJ,
    2. Furger A,
    3. Dye MJ
    (2002) Integrating mRNA processing with transcription. Cell 108:501–512.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Andrecka J,
    2. et al.
    (2008) Single-molecule tracking of mRNA exiting from RNA polymerase II. Proc Natl Acad Sci USA 105:135–140.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Liphardt J,
    2. Onoa B,
    3. Smith SB,
    4. Tinoco IJ,
    5. Bustamante C
    (2001) Reversible unfolding of single RNA molecules by mechanical force. Science 292:733–737.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Abels JA,
    2. Moreno-Herrero F,
    3. van der Heijden T,
    4. Dekker C,
    5. Dekker NH
    (2005) Single-molecule measurements of the persistence length of double-stranded RNA. Biophys J 88:2737–2744.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Gu W,
    2. Reines D
    (1995) Identification of a decay in transcription potential that results in elongation factor dependence of RNA polymerase II. J Biol Chem 270:11238–11244.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Kim HD,
    2. et al.
    (2002) Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc Natl Acad Sci USA 99:4284–4289.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Chung WH,
    2. et al.
    (2003) RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol Cell 12:1003–1013.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Wade PA,
    2. et al.
    (1996) A novel collection of accessory factors associated with yeast RNA polymerase II. Protein Expr Purif 8:85–90.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Okten Z,
    2. Churchman LS,
    3. Rock RS,
    4. Spudich JA
    (2004) Myosin VI walks hand-over-hand along actin. Nat Struct Mol Biol 11:884–887.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Churchman LS,
    2. Okten Z,
    3. Rock RS,
    4. Dawson JF,
    5. Spudich JA
    (2005) Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc Natl Acad Sci USA 102:1419–1423.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Radman-Livaja M,
    2. Biswas T,
    3. Mierke D,
    4. Landy A
    (2005) Architecture of recombination intermediates visualized by in-gel FRET of λ integrase–Holliday junction–arm DNA complexes. Proc Natl Acad Sci USA 102:3913–3920.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Ha T,
    2. et al.
    (1999) Ligand-induced conformational changes observed in single RNA molecules. Proc Natl Acad Sci USA 96:9077–9082.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Rasnik I,
    2. McKinney SA,
    3. Ha T
    (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893.
    OpenUrlCrossRefPubMed
  50. ↵
    1. DeLano WL
    (2002) The PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, CA).
  51. ↵
    1. Jones TA,
    2. Zou JY,
    3. Cowan SW,
    4. Kjeldgaard M
    (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47:110–119.
    OpenUrlCrossRefPubMed
PreviousNext
Back to top
Article Alerts
Email Article

Thank you for your interest in spreading the word on PNAS.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis
(Your Name) has sent you a message from PNAS
(Your Name) thought you would like to see the PNAS web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis
Chin-Yu Chen, Chia-Chi Chang, Chi-Fu Yen, Michael T.-K. Chiu, Wei-Hau Chang
Proceedings of the National Academy of Sciences Jan 2009, 106 (1) 127-132; DOI: 10.1073/pnas.0811689106

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis
Chin-Yu Chen, Chia-Chi Chang, Chi-Fu Yen, Michael T.-K. Chiu, Wei-Hau Chang
Proceedings of the National Academy of Sciences Jan 2009, 106 (1) 127-132; DOI: 10.1073/pnas.0811689106
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Mendeley logo Mendeley

Article Classifications

  • Biological Sciences
  • Biophysics
Proceedings of the National Academy of Sciences: 106 (1)
Table of Contents

Submit

Sign up for Article Alerts

Jump to section

  • Article
    • Abstract
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & SI
  • Info & Metrics
  • PDF

You May Also be Interested in

Water from a faucet fills a glass.
News Feature: How “forever chemicals” might impair the immune system
Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
Image credit: Shutterstock/Dmitry Naumov.
Reflection of clouds in the still waters of Mono Lake in California.
Inner Workings: Making headway with the mysteries of life’s origins
Recent experiments and simulations are starting to answer some fundamental questions about how life came to be.
Image credit: Shutterstock/Radoslaw Lecyk.
Cave in coastal Kenya with tree growing in the middle.
Journal Club: Small, sharp blades mark shift from Middle to Later Stone Age in coastal Kenya
Archaeologists have long tried to define the transition between the two time periods.
Image credit: Ceri Shipton.
Mouse fibroblast cells. Electron bifurcation reactions keep mammalian cells alive.
Exploring electron bifurcation
Jonathon Yuly, David Beratan, and Peng Zhang investigate how electron bifurcation reactions work.
Listen
Past PodcastsSubscribe
Panda bear hanging in a tree
How horse manure helps giant pandas tolerate cold
A study finds that giant pandas roll in horse manure to increase their cold tolerance.
Image credit: Fuwen Wei.

Similar Articles

Site Logo
Powered by HighWire
  • Submit Manuscript
  • Twitter
  • Facebook
  • RSS Feeds
  • Email Alerts

Articles

  • Current Issue
  • Special Feature Articles – Most Recent
  • List of Issues

PNAS Portals

  • Anthropology
  • Chemistry
  • Classics
  • Front Matter
  • Physics
  • Sustainability Science
  • Teaching Resources

Information

  • Authors
  • Editorial Board
  • Reviewers
  • Subscribers
  • Librarians
  • Press
  • Cozzarelli Prize
  • Site Map
  • PNAS Updates
  • FAQs
  • Accessibility Statement
  • Rights & Permissions
  • About
  • Contact

Feedback    Privacy/Legal

Copyright © 2021 National Academy of Sciences. Online ISSN 1091-6490