Consistences of individual DNA base—amino acid interactions in structures and sequences

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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“…*Values an average of at least two measurements. side chains, and T bases are more frequently contacted by Gln than Arg side chains (33).…”

Section: Resultsmentioning

confidence: 99%

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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“…It has been shown that the charged residues play a major role in the protein-nucleotide interactions [26][27][28]. We proceed to study the effect of this charge-charge interaction on translocation time.…”

Section: United Atom Modelmentioning

confidence: 99%

Modeling of polynucleotide translocation through protein pores and nanotubes

Kong¹,

Muthukumar²

2002

Electrophoresis

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In an effort to understand recent experiments, we have performed Brownian dynamics simulations of polymer translocation through nanometer-scale protein pores under the influence of an external applied electric field. Multiple peaks in the translocation time distribution are observed in agreement with experiments. Under the same conditions, but replacing the protein pore with a rigid cylindrical tube of comparable size, only a single peak is observed in the translocation time distribution. These results directly show that the geometry of the protein pores is mainly responsible for multiple peaks observed in experiments. In the case of alpha-hemolysin channel, we find the vestibule, by confining many conformations of the translocating polymer, to be responsible for the second peak with longer translocation time.

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“…It seems reasonable to think that residues Arg-265 and Asn-268 mediate base-specific or nonspecific interactions with the DNA backbone, because arginine and asparagine are known to participate in such interactions (48). Since Leu-262 may comprise part of the ␣CTD hydrophobic core, the Leu to Ala substitution may lead to an altered conformation in which the presentation of residues directly contacting DNA and/or Mor is affected, resulting in reduced transcription activation.…”

Section: Fig 5 Interaction Trap Assaymentioning

confidence: 99%

Transcription Activation by the Bacteriophage Mu Mor Protein Requires the C-terminal Regions of Both α and σ70 Subunits of Escherichia coli RNA Polymerase

Artsimovitch

Murakami

Ishihama

et al. 1996

Journal of Biological Chemistry

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Middle transcription of bacteriophage Mu requiresEscherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, P m , has a ؊10 hexamer but lacks a recognizable ؊35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in ␣ or 70 . As predicted if ␣ were binding to P m , we detected a polymerase-dependent footprint in the ؊60 region. Reconstituted RNA polymerases containing Ala substitutions in the ␣ C-terminal domain were used to assay Mor-dependent transcription from P m in vitro. The D258A substitution and ␣ deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both ␣ and 70 ; consistent with a key role of ␣-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) ␣-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via ␣-DNA contact.Bacteriophage Mu is a temperate phage of Escherichia coli K-12 and several other enteric bacteria (1). Mu uses the host RNA polymerase (RNAP) 1 throughout its lytic development (2) to transcribe three sets of genes: early, middle, and late (3). Middle operon transcription requires phage DNA replication and the early gene product Mor (3, 4) and results in expression of C, which in turn activates transcription of late genes encoding phage morphogenesis, cell lysis, and DNA modification functions (5, 6). Transcription from the middle promoter, P m , requires activation by Mor and is carried out by the E. coli RNAP holoenzyme containing 70 (4, 7). The detailed mechanism by which this activation occurs remains unknown; for example, it might involve protein-protein interactions between Mor and RNA polymerase, conformational changes in the promoter DNA, or a combination of both.Previous in vivo and in vitro footprinting analysis of P m revealed single-stranded bases resulting from distortion in the Ϫ33 region, close to the predicted interface between Mor and RNAP (8). The distortion was dependent on the presence of both Mor and RNAP in vitro and involved strand separation confined to positions Ϫ35 through Ϫ31, as inferred from sensitivity to KMnO 4 modification and Mung bean or S1 nuclease cleavage following modification with dimethyl sulfate. This unwinding was enhanced or abolished in Up or Down spacerregion mutants, respectively, indicating that it may play a role in the activation of transcription.The middle promoter possesses characteristic features of a promoter under positive control (9) (Fig. 1); it has a recognizable Ϫ10 hexamer but lacks similarity to the canonical Ϫ35 hexamer (at most, a 2-base pair match to consen...

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Consistences of individual DNA base—amino acid interactions in structures and sequences

Cited by 46 publications

References 14 publications

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

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

Modeling of polynucleotide translocation through protein pores and nanotubes

Transcription Activation by the Bacteriophage Mu Mor Protein Requires the C-terminal Regions of Both α and σ70 Subunits of Escherichia coli RNA Polymerase

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