Crystal Structure of T7 Gene 4 Ring Helicase Indicates a Mechanism for Sequential Hydrolysis of Nucleotides

Singleton, Martin R.; Sawaya, M.R.; Ellenberger, Tom; Wigley, Dale B.

doi:10.1016/s0092-8674(00)80871-5

Cited by 454 publications

(565 citation statements)

References 49 publications

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“…We propose that the side chain of Asn 149 contributes to the catalytic site geometry that allows for the high turnover rates of PspF. Our functional assignment is in agreement with the previously suggested roles of the second sensor I residue in other AAAϩ proteins (37,38).…”

Section: The Asparagine Of the Pspf Sensor I Is Involved In Atp Hydrosupporting

confidence: 90%

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Schumacher

Joly

Rappas

et al. 2007

Journal of Biological Chemistry

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Transcriptional initiation invariably involves the transitionRegulation of transcription enables cells to adapt and differentiate through coordination of protein synthesis. Two major mechanisms exist to control gene transcription as follows: one through recruitment to or preventing the RNA polymerase (RNAP) 4 binding to promoter DNA and one through activation or inhibition of the RNA polymerase activity. Despite the variations in subunit numbers that constitute the basal transcription machinery, RNA polymerases are structurally and functionally highly conserved in all kingdoms of life (1). During the initiation of transcription, RNA polymerases invariably melt the promoter DNA and have to engage the single-stranded template DNA in the structurally conserved catalytic cleft (2).The major variant bacterial 54 -RNAP is regulated by an activating mechanism with some resemblance to the operation of eukaryotic RNAP II. In both cases, passing from the closed to the open RNAP promoter complex requires activator proteins that hydrolyze nucleoside triphosphate to drive open RNAP promoter complex formation (3). The 54 -RNAP binds to specific promoter sites centered on positions Ϫ24 and Ϫ12 relative to the transcription start site and remains in a transcriptionally silent conformation. Open complexes of the 54 -RNAP promoter complexes are thermodynamically labile, and activation relies on the productive coupling of nucleoside triphosphate hydrolysis (energy coupling) from 54 activators to the 54 -RNAP promoter complex. The 54 activator-dependent initial events in open complex formation are mediated by structural changes between 54 and the promoter DNA, which can be melted from position Ϫ12 to Ϫ5 in a reaction that can occur independently from RNAP core determinants. The restructuring of promoter DNA by activated 54 has been termed 54 isomerization (4 -6). Following the initial events and requiring the presence of the RNAP core, the promoter DNA opening extends to position ϩ3 relative to the transcription start site (7).Energy coupling primarily involves intimate interactions between the activator and the 54 promoter complex. 54 activators, also termed enhancer-binding proteins (EBP), belong to the versatile AAAϩ protein (ATPases associated with various cellular activities) family of molecular machines (for review see Refs. 8 -10). Members of EBPs include the well studied NtrC, PspF, DctD, XylR, NifA, and DmpR proteins (11). PspF is the phage shock protein F that activates transcription of psp genes involved in the phage shock response in Escherichia coli (12, * This work was supported in part by the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. Tel.: 44-207-5945366; Fax: 44-207-5842056; E-mail: j.schumacher@imperial.ac.uk. 2 Recipient of EMBO F...

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Section: The Asparagine Of the Pspf Sensor I Is Involved In Atp Hydrosupporting

confidence: 90%

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Schumacher

Joly

Rappas

et al. 2007

Journal of Biological Chemistry

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“…Therefore, we propose a model in which TrwB operates as a molecular motor, translocating DNA by a mechanism similar to that used by F 1 -ATPase. A somehow similar mechanism has been proposed for DNA movement across T7 gene4 hexameric helicase (10,27).…”

Section: Discussionmentioning

confidence: 55%

TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase

Tato

Zunzunegui

Cruz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

104

122

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Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwB⌬N70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwB⌬N70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwB⌬N70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwB⌬N70 -DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F 1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor. molecular motor ͉ DNA transfer C onjugative plasmids provide a main route for acquisition of new genetic information in bacteria. Bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show similarities to both DNA replication and protein transport systems (1, 2). The translocated substrate is a nucleoprotein particle that crosses the bacterial envelope into other bacterial or eukaryotic cells, crossing the kingdom boundaries. A two-step mechanism for DNA transport was proposed (2), in which conjugation is visualized as a DNA rolling-circle replication process linked to a type IV protein secretion system. Conjugation is encoded by two gene clusters in most conjugative plasmids. One cluster (dtr) encodes the DNA transfer replication proteins, and the other (mpf ) codes for the proteins that assemble the type IV protein secretion system channel. R388 is a 34-kb plasmid, which displays the simplest known dtr region, encoding only three proteins (TrwA, TrwB, and TrwC). The mpf region codes for 11 proteins (TrwD-N) involved in the formation of an extracellular pilus and a membrane-associated complex that translocates the nucleoprotein substrate across membranes.TrwB, an integral membrane and DNA-binding protein, is a key player in R388 conjugation (3-6). It has a counterpart in all conjugative systems, and it is thought to be responsible for recruiting the relaxosome DNA-pr...

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“…This result suggests that hetero-oligomers are formed and that the presence of a wild-type linker region can dissipate the extensive oligomerization. These results are interesting in view of the earlier finding that a T7 gene 4 helicase domain having a deletion in this portion of the linker region formed extended oligomers in the crystal structure rather than hexamers (9) whereas the helicase domains containing this portion of the linker region crystallized as hexamers (10).…”

Section: T7 Helicase-primase Linker Regionmentioning

confidence: 68%

“…Biochemical data suggested that this linker region is critical for the functioning of the gene 4 protein (8). As anticipated from the biochemical data, crystal structures of the helicase domains revealed residues in the linker of one subunit of the hexamer contacting residues in the adjacent subunit, thus contributing to the stability of the hexamer (9,10). Although the structure showed clearly the participation of the linker in subunit interaction, 9 of 26 residues were disordered in the structure.…”

Section: Screening Of Permutable Amino Acid Changes At Specific Positmentioning

confidence: 71%

“…Helicase fragments lacking this linker region failed to oligomerize except at extremely high protein concentration and were severely impaired for all helicase activities (8). The crystal structure of the hexameric helicase fragment subsequently revealed that the subunit interface of the helicase ring is stabilized by interactions between an N-terminal region (residues 264 -284 or helix A) of one subunit and a pocket on the adjacent subunit (residues 364 -395 or helices D2 to D3) (10) (Fig. 1).…”

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confidence: 97%

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The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

Lee

Richardson

2004

Journal of Biological Chemistry

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The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for templatedirected oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246 -271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala 257 , Pro 259 , and Asp 263 ) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala 257 and Asp 263 to be essential whereas Pro 259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala 257 or Asp 263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg 2؉ , all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.

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Crystal Structure of T7 Gene 4 Ring Helicase Indicates a Mechanism for Sequential Hydrolysis of Nucleotides

Cited by 454 publications

References 49 publications

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase

The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

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