Activity of coliphage HK022 excisionase (Xis) in the absence of DNA binding.
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A mutated excisionase (Xis) protein of coliphage HK022 whose single Cys residue was replaced by Ser does not bind to its two tandem binding sites (X1, X2) on the P arm of attR. Despite its DNA-binding inability the protein showed 30% excision activity of the wild type Xis both in vitro and in vivo. This partial activity is attributed to the interaction of Xis with integrase that is retained in the mutant protein. This protein-protein interaction occurs in the absence of DNA binding. (+info)
Suppression of factor-dependent transcription termination by antiterminator RNA.
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Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put. put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator. (+info)
Solution structure and stability of the full-length excisionase from bacteriophage HK022.
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Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins. (+info)
Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues.
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The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH(2)-terminal arginine-rich motif binds BOXB sequence in nascent lambda transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp(108) is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys(106) and Lys(107) residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude lambda. All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/K107R and K106A/K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys(106) and Lys(107) and negatively charged DNA phosphate groups. These may assist intercalation of Trp(108) into template. (+info)
Phosphorylation of the integrase protein of coliphage HK022.
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