pubmed.ncbi.nlm.nih.gov

Running in reverse: the structural basis for translocation polarity in hexameric helicases - PubMed

  • ️Thu Jan 01 2009

Running in reverse: the structural basis for translocation polarity in hexameric helicases

Nathan D Thomsen et al. Cell. 2009.

Abstract

Hexameric helicases couple ATP hydrolysis to processive separation of nucleic acid duplexes, a process critical for gene expression, DNA replication, and repair. All hexameric helicases fall into two families with opposing translocation polarities: the 3'-->5' AAA+ and 5'-->3' RecA-like enzymes. To understand how a RecA-like hexameric helicase engages and translocates along substrate, we determined the structure of the E. coli Rho transcription termination factor bound to RNA and nucleotide. Interior nucleic acid-binding elements spiral around six bases of RNA in a manner unexpectedly reminiscent of an AAA+ helicase, the papillomavirus E1 protein. Four distinct ATP-binding states, representing potential catalytic intermediates, are coupled to RNA positioning through a complex allosteric network. Comparative studies with E1 suggest that RecA and AAA+ hexameric helicases use different portions of their chemomechanical cycle for translocating nucleic acid and track in opposite directions by reversing the firing order of ATPase sites around the hexameric ring. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Rho/RNA/ADP•BeF3•Mg2+ structure

(A) Top down view of the Rho hexamer with differential coloring of the six subunits (see color key). Bound RNA is shown as orange sticks. ADP, BeF3 and Mg2+ are shown as magenta sticks, black sticks, and yellow-green spheres respectively. (B) Side view of Rho rotated 90° with respect to (A), and shown with a transparent surface. Subunits A and B have been removed to highlight the RNA. (C) Representative Fo−Fc electron density in the ATPase active site calculated prior to including nucleotide in the model (3 σ contouring). The refined ADP•BeF3•Mg2+ model is shown to highlight the starting map quality. (D) Representative Fo−Fc electron density (green) for RNA calculated prior to including the nucleic acid substrate in the model (left, 2.5 σ contouring), and refined 2Fo−Fc electron density (blue) for the final model (right, 1.25 σ contouring). The refined RNA model is shown in orange with oxygen and nitrogen atoms colored red and blue, respectively.

Figure 2
Figure 2. Translocation loop/RNA interactions

(A) The Rho Q loops (left) and E1 β-hairpin loops (right) bind the nucleic acid backbone in a spiral staircase configuration. Both sets of nucleic acid binding loops are structured in a manner suggesting that they pull (arrows), rather than push, on the substrate to achieve a particular translocation polarity (5’→3’ for Rho vs. 3’→5’ for E1). The Rho Q loops for subunits A–E are shown (see key color key, panel B); subunit F, which does not contact the RNA, is removed for clarity. The E1 loops are colored similarly to those in Rho based on their position in the staircase. Nucleic acids are colored orange and illustrated as sticks on a cartoon backbone. The orientation of the Rho hexamer is similar to that shown in Figure 1B. (B) The Rho R loops form a spiral staircase that engages the RNA phosphates. The R loops for all six subunit are colored (see key). The 5’ end of the RNA (orange and red sticks) projects out, toward the viewer. The side-chains of K326 (sticks), project into the center of the ring and interact with the RNA phosphate groups. (C) Specific RNA contacts with the Q and R loops of subunits B and C (see color key, panel B). Backbone atoms and side-chains for the RNA-binding residues are shown as sticks. Dashed lines indicate hydrogen-bonds or salt bridges.

Figure 3
Figure 3. Structural asymmetry generates four ATP binding states

Views of (A) Exchange - E; (B) ATP - T; (C) ATP hydrolysis - T*; (D) ADP - D active sites. Each inset shows the interface location (bold and black text) with respect to the rest of the hexamer (faded and grey text). For nucleotide and associated Mg2+/water molecules, coloring is by B-factor, with blue indicating low values (20–35 Å2) and red high values (120–135 Å2). By contrast, selected catalytic motifs are shown in stick representation and colored by subunit in accordance with Figure 1A. ADP and BeF3 are shown as sticks. Bound Mg2+ and associated water molecules are shown as large and small spheres, respectively; the water molecules are numbered according to their position in the active site. Bonding interactions are shown as dashed lines. Abbreviations: WA – Walker A, WB – Walker B, CE – Catalytic Glutamate, RV – Arginine Valve, RF – Arginine Finger.

Figure 4
Figure 4. Correlation between ATP and RNA binding

Schematized view of RNA-binding contacts and ATP-binding states. Protein subunits are illustrated as large, rounded rectangles and colored as per Figure 1A. Q and R loops are drawn with darker, colored lines to highlight their positions. The perspective is similar to Figure 1B, except that the subunits are pulled open and spread flat on the page. Subunit F is shown twice to highlight its orientation with respect to subunits A and E. The two halves of the bipartite ATP-binding site are illustrated as small rounded rectangles; linked, notched rectangles represent insertion of the arginine finger into the active site of T and T* states. Ribose (R) and phosphate (P) moieties of the RNA backbone are colored orange and numbered according to the structure. Protein residues contacting RNA are labeled, and chemical groups that bond with the RNA are shown (dashed lines). The yellow star indicates the B/C interface in which adjacent subunits have maximized their protein-RNA contacts.

Figure 5
Figure 5. Inter- and intra-subunit conformational changes position the catalytic glutamate via a conserved allosteric network

(A) Inter-subunit conformational changes in Rho. Three different interfaces (F/A, E/F, B/C) are shown. The inset identifies the subunits and active site states represented by each interface. The product-release (D) and exchange (E) states are relatively open (i.e. more accessible to solvent), whereas the ATP bound (T and T*) states (illustrated here by the B/C interface) are closed (sequestered from solvent). These changes alter the relative positions of adjacent Q and R loops and their associated secondary structural elements, Rα1, Qα1 and Qα2, which are labeled and colored by subunit (see key, panel B). Selected residues involved in inter-subunit contacts are labeled and shown as sticks, and bonding interactions as dashed lines. (B) Intra-subunit conformational changes illustrated by structural superposition of the RecA-folds from all six subunits. The core ASCE fold is colored white, while the Q and R loops, and the Rα1, Qα1 and Qα2 structural elements are colored by subunit (see key). While most of the motor domain shows minimal intra-subunit conformational changes, the RNA binding elements exhibit significant variations. (C) The position of the allosteric network with respect to RNA (orange cartoon) and the presence of a prospective catalytic water molecule (large red spheres) in the ATPase active site. The Rα1, Qα1 and Qα2 structural elements encircle the RNA binding site and form a large portion of the subunit interface within the hexamer. Charged residues (sticks) project from Rα1, Qα1 and Qα2, forming a network between subunits. Bonding interactions (dashed lines) within the network vary around the hexamer depending on nucleotide state. Protein subunits are differentially colored (see key, panel B). (D) The completely bonded allosteric network located at the T* interface between subunits B and C. The inter-subunit conformational changes shown in panel A are linked to an interaction between R347 and E333. All members of the network except R347 project from the Rα1, Qα1 and Qα2 structural elements of adjacent subunits and form a complex network of salt bridges that affects the position of the catalytic glutamate (E211B) responsible for activating the nucleophilic water molecule. ADP and BeF3 are colored as magenta and black sticks. Protein side-chains are illustrated as sticks and colored by subunit (see key, panel B). The Mg2+ ion and catalytic water molecule are shown as yellow-green and red spheres, respectively. See also supplemental movie S1 and movie S2.

Figure 6
Figure 6. Translocation mechanism and directional polarity

(A) Schematic of a Rho translocation cycle in which six ATP molecules are hydrolyzed to move six nucleotides of RNA. Helicase subunits are illustrated as colored spheres. RNA is shown as a chain of white spheres spiraling out of the plane of the paper. Protein-RNA contacts are indicated by lines connecting the protein and RNA spheres; the black RNA sphere serves as a reference point, and moves toward the viewer as the boxed red subunit transitions through six steps in the translocation cycle. A yellow star represents activation of the allosteric network that likely promotes hydrolysis. See also supplemental movie S3–movie S6. (B) Schematics of Rho and E1 (chains A–F) illustrating their respective sequential ATP hydrolysis directions. Protein subunits are colored as in Figure 1. Nucleic acid phosphates observed in the structures are illustrated as bold orange circles, with the incoming phosphate shown as a dashed orange circle. Rectangles represent the two halves of the bipartite active site. Interlocked rectangles show insertion of the arginine finger in ATP-bound states. Solid arrows outline the progression toward subsequent steps in the ATPase cycle. Dotted arrows show the movement of the mobile “transition” subunit, upon binding ATP, toward a partner subunit locked in place by ATP-dependent (T-state) contacts within the ring.

Comment in

Similar articles

Cited by

References

    1. Abrahams JP, Leslie AG, Lutter R, Walker JE. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 1994;370:621–628. - PubMed
    1. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr. 2002;58:1948–1954. - PubMed
    1. Adelman JL, Jeong YJ, Liao JC, Patel G, Kim DE, Oster G, Patel SS. Mechanochemistry of transcription termination factor Rho. Mol Cell. 2006;22:611–621. - PubMed
    1. Berger JM. SnapShot: nucleic acid helicases and translocases. Cell. 2008;134:888–888. e881. - PMC - PubMed
    1. Brennan CA, Dombroski AJ, Platt T. Transcription termination factor rho is an RNA-DNA helicase. Cell. 1987;48:945–952. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources