Subdomain organization and catalytic residues of the F factor TraI relaxase domain

Street, Lara; Harley, Matthew J.; Stern, J. C.; Larkin, Chris; Williams, S.; Miller, Dana L.; Dohm, Julie A.; Rodgers, Michael E.; Schildbach, Joel F.

doi:10.1016/s1570-9639(02)00553-8

Cited by 42 publications

(64 citation statements)

References 43 publications

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“…It is known that the transesteriWcation reaction carried out by all relaxases characterized thus far is dependent on the action and proximity of 3 or 4 key residues at the active site, one of which is the absolutely conserved catalytic tyrosine residue required for the formation of a covalent phosphotyrosyl bond between DNA and protein (Larkin et al, 2005;Pansegrau et al, 1994;Scherzinger et al, 1993;Street et al, 2003). It is expected that the relaxase encoded by R. erythropolis AN12 pREA400 traA will function similarly because these residues appear to be conserved (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, this region is 27% identical to TraA relaxase/helicase encoded by the Agrobacterium tumefaciens pTiC58 plasmid (Farrand et al, 1996), and 22% identical to the TraI relaxase/helicase of the E. coli F factor (AbdelMonem et al, 1983;Larkin et al, 2005;Street et al, 2003). Alignments to the consensus helicase motifs (Hall and Matson, 1999) revealed that pREA400 TraA and its actinomycete relatives belong to the superfamily I (SFI) type of helicases (Fig.…”

Section: Sequence Analysis Of Prea400 Encoded Traamentioning

confidence: 99%

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TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

Yang

Lessard

Sengupta

et al. 2007

Plasmid

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Pulsed-Weld gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100 kb, respectively, based on their migration in pulsed-Weld gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7 £ 10 ¡4 and 5 £ 10 ¡4 events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we conWrmed that the conjugation defect was speciWcally due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.

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Section: Discussionmentioning

confidence: 99%

Section: Sequence Analysis Of Prea400 Encoded Traamentioning

confidence: 99%

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

Yang

Lessard

Sengupta

et al. 2007

Plasmid

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“…All cloned gene segments and mutations were confirmed by DNA sequencing. The F TraI36 expression construct (pET24a-traI36) was engineered previously (27). For R100 TraI36 expression, the N-terminal 331-codon region of R100 traI was PCR amplified.…”

Section: Methodsmentioning

confidence: 99%

“…The R100 traI36 gene was excised from pNEB193, ligated into NdeI͞EcoRI-digested pET24a(ϩ) (Novagen), and transformed into strain XL10 Gold. The 3Ј-most codon was removed by PCR as described (27).…”

Section: Methodsmentioning

confidence: 99%

“…We previously demonstrated, by using the 36-kDa relaxase domain of F TraI (TraI36), an N-terminal 330-aa fragment of F factor TraI (27), that this specificity can be remarkable. F TraI36 binds a single-stranded F oriT sequence with a subnanomolar K D , and some single-base substitutions over a 10-base region can reduce affinity by between 10-and 10,000-fold (24).…”

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confidence: 99%

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Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Harley

Schildbach

2003

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

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Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand siteand sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R͞R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q͞Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution.

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An Engineered DNA‐Binding Protein Self‐assembles Metallic Nanostructures

Sedlak¹,

Hnilova²,

Gachelet³

et al. 2010

ChemBioChem

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Biological fabrication routes can provide a way to overcome the limitations presented by current chemistry-based nanoparticle arrangement and assembly methods. Many recent assembly strategies utilize DNA as the templating molecule by patterning gold nanoparticles on DNA through chemical conjugation via, for example, a sulfhydryl bond.[1] Reliance upon this chemistry, however, limits applications because it acts indiscriminately on several different metals and is only useful for some noble-metal nanoparticles. Strategies that covalently link nanoparticles to proteins or DNA risk denaturation or distortion of native protein, distortion of DNA, and/or disruption of the plasmonic or photonic properties unique to nanoparticles.[2] We present a strategy for nanoparticle patterning on DNA that utilizes the biologically based self-assembly properties of DNA-binding proteins to facilitate the targeted immobilization of nanoparticles on DNA. Here we show that a derivative of the DNA-binding protein TraI spontaneously organizes colloidal gold nanoparticles on DNA through an engineered gold-binding peptide motif. This system, based solely on specific, noncovalent, biologically determined interactions, represents significant progress on the route to spontaneously ordered assembly of nanoparticles important for downstream applications in nanoelectronics and photonics.TraI (192 kDa, 1756 residues) is the E. coli F-plasmid-encoded relaxase/helicase that harbors sequence-specific single-stranded-DNA-binding activity (relaxase domain) and nonspecific single and double-stranded-DNA-binding activity (helicase domain).[3] We engineered TraI with a gold-binding motif at an internal permissive site after residue Q369 to direct the assembly of gold nanoparticles (AuNPs) on DNA through noncovalent interactions. Permissive sites, regions of proteins that tolerate a wide variety of amino acid additions without disrupting native protein function, were previously identified through transposon/epitope tag mutagenesis in TraI. [4,5] This study utilizes TraI's nonspecific DNA-binding activity as the first step toward optimization of this biologically based nanoparticletemplating strategy. Because TraI is also capable of sequencespecific DNA binding, this work paves the path to the final step of the biologically based strategy: addressable, targeted immobilization of nanoparticles on DNA.Inorganic binding peptides identified by several groups [6,7]

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Subdomain organization and catalytic residues of the F factor TraI relaxase domain

Cited by 42 publications

References 43 publications

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

An Engineered DNA‐Binding Protein Self‐assembles Metallic Nanostructures

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