Saturday, 16 June 2012

Running in Reverse: The Structural Basis for Translocation Polarity in Hexameric Helicases by Thomsen and Berger


Introduction

  • 2 major families of hexameric helicases
  • Euk, euk viruses and archae use AAA+ enz
  • translocate 3' --->5' along substrates
  • bacteria and their phages use RecA-type helicases eg DnaB and  T7gp4
  • move 5' ---> 3' 
  • bac Rho factor is a RecA-family heexameric helicase
  • terminate transcrip at discrete genomic loci
  • control gene expression
  • Rho loads onto nascent RNA strands at rut sites
  • with primary, cytosine-spec RNA binding activity
  • Loading lets RNA interact with a seconday binding site in central channel of hexmaer
  • stimulates an RNA-dependent ATPase activity
  • coupling ATP turnover to secondary-site binding thought to translocate Rho 5' ---> 3' along RNA towards a transcribing polymerase
  • on reaching transcription complex, Rho may forcibly dissoc a paused RNAP
  • by using its motor activity to separate RNA-DNA heteroduplex or pushing pol forwards to collapse transcription bubble

To examine these issues, we determined the structure of a Rho hexamer bound to the ATP mimic ADP•BeF3and a centrally bound RNA oligonucleotide. Six bases of RNA are coordinated by a spiral staircase of loops that form contacts with the nucleic acid backbone. RNA binding coincides with the formation of an asymmetric particle that contains four distinct classes of ATP-binding sites, which together appear to recapitulate different catalytic states consistent with a sequential ATP hydrolysis mechanism. Comparison of Rho and E1 hexamers reveals that the two motors bind nucleic acid substrates in a similar conformation and with the same relative polarity (Enemark and Joshua-Tor, 2006), but that different ATPase states are responsible for DNA or RNA binding. These features suggest that Rho and E1 translocate in opposite directions because the sequential firing orders of their respective ATPase sites may be reversed.
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 Movies S3–S6.(B) Schematics of Rho and E1 (chains A–F) illustrating their respective sequential ATP hydrolysis directions. Protein subunits are colored as inFigure 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.
  • during translocation each Rho subunit is thought to transition thru a round of ATP binding, hydrolysis and product release
  • STr suggests Rho uses an   [E → T1 → T2 → T1 → T2 → D] ATPase cycle
  • full circuit involves ADP release and binding a new ATP at lone E state
  • progressively tighter binding of ATP in T states
  • ATP hydrolysis in one of the T* states
  • stable product formation in D state
  • hydrolysis likely occurs in allosterically activated T*1 state
  • Pi release delayed until partially open D or fully open E is reached
  • subunits change relative conform with respect to each other in closed, cyclic wave
  • pulls RNA thru central channel of helicase
  • As ATP is bound by an E-state subunit, protomer latches onto an incoming 3'-RNA nt
  • As subunit transitions into T and then T* states, it chaperones that nt up thru ring
  • as RNA prepares to exit Rho, str changes in Q loop reduce number of protein-RNA contacts
  • prime nucleic acid for release
  • to complete a cycle, subunit enters D state
  • transition from top to bottom of staircase
  • diengages from RNA
  • str support sequential rotary ATP hydrolysis
  • each ATP turnover translocates motor by 1 RNA base
  • in Rho, allosteric network linking ATP- and RNA-binding sites is acticated by tight binding of RNA to a pair of adj subunits
  • explains how Rho can bind multiple ATP mols tightly and hydrolyse them in an ordered, sequential manner rather than concerted fashion
  • hexamer is a particle in wh 5 subunits simultaneously contact nucleic acid
  • a single subunit stays assoc with 1 RNA mol thruout catalytic cycle

RecA and AAA+ Hexameric Helicases: The Structural Basis for Translocation Polarity

  • RecA-family enz move 5' ---> 3'
  • AAA+ proteins track 3' ---> 5'
  • each hexamer's ATP-binding sites are formed with Walker A/B motifs counterclockwise from neighbouring arginine finger
  • helicase contain an exchange/ empty (E) ATP-binding site sequestered between a tightly bound ATP (T) state and a weakly bound product (D) state
  • protomer may be linked to T state effectively being locked down
  • subunit between E and D states free to move on ATP binding
  • inverted orientation leads to CCW shift of free subunit on binding ATP in Rho and CW shift in E1



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