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

Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2003; 100(20):11243-8. DOI: 10.1073/pnas.2035001100
Source: PubMed


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 site- and 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 Q193R/R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q/Q201R) 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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    • "This selectivity is largely due to the interactions of a non-conserved pair of amino acid residues , Gln193 and Arg201 in F TraI, and a pair of singlestranded bases at 145′ and 147′ (according to the basenumbering scheme of the nic site in Frost et al., 1994) (Fig. 3C). The specificity of binding can be swapped to some extent between R100 and F by switching residues only at these positions (Harley and Schildbach, 2003). "
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    ABSTRACT: The tra operon of the prototypical F plasmid and its relatives enables transfer of a copy of the plasmid to other bacterial cells via the process of conjugation. Tra proteins assemble to form the transferosome, the transmembrane pore through which the DNA is transferred, and the relaxosome, a complex of DNA-binding proteins at the origin of DNA transfer. F-like plasmid conjugation is characterized by a high degree of plasmid specificity in the interactions of tra components, and is tightly regulated at the transcriptional, translational and post-translational levels. Over the past decade, X-ray crystallography of conjugative components has yielded insights into both specificity and regulatory mechanisms. Conjugation is repressed by FinO, an RNA chaperone which increases the lifetime of the small RNA, FinP. Recent work has resulted in a detailed model of FinO/FinP interactions and the discovery of a family of FinO-like RNA chaperones. Relaxosome components include TraI, a relaxase/helicase, and TraM, which mediates signalling between the transferosome and relaxosome for transfer initiation. The structures of TraI and TraM bound to oriT DNA reveal the basis of specific recognition of DNA for their cognate plasmid. Specificity also exists in TraI and TraM interactions with the transferosome protein TraD.
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    • "The amino acid exchanges introduced to TraI N1-992 were chosen because these are known to substantially reduce the affinity of the protein for sequences surrounding nic (Harley and Schildbach, 2003). Reduced affinity of the relaxase-associated binding site was confirmed for a truncated TraI N1-330 M2L/E153D/Q193R/R201Q (K. "
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    • "The situation, however, can complicate analysis when examining the effects of amino acid substitutions. As part of a mutagenesis study designed to examine the contributions of various amino acids to TraI36 DNA recognition, we generated a series of protein variants with Ala substituting for contact residues (Harley and Schildbach, 2003; Larkin et al., 2005). Usually, the effects of variant proteins on anisotropy and intensity of a 3′-TAMRA-labeled 22-base oligonucleotide were similar to those caused by binding of the wild-type protein. "
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    ABSTRACT: Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with a variety of labeled oligonucleotides and the relaxase domain of F plasmid TraI. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.
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