P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities.
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The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles. (+info)
Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.
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The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda. Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees. Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase. (+info)
In vitro selection of integration host factor binding sites.
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Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function. (+info)
Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.
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The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies. (+info)
Activation of Escherichia coli leuV transcription by FIS.
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The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription. (+info)
Site-specific recombination of bacteriophage P22 does not require integration host factor.
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Site-specific recombination by phages lambda and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates lambda site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting lambda site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes. (+info)
RpoS synthesis is growth rate regulated in Salmonella typhimurium, but its turnover is not dependent on acetyl phosphate synthesis or PTS function.
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The RpoS sigma factor of enteric bacteria is either required for or augments the expression of a number of genes that are induced during nutrient limitation, growth into stationary phase, or in response to stresses, including high osmolarity. RpoS is regulated at multiple levels, including posttranscriptional control of its synthesis, protein turnover, and mechanisms that affect its activity directly. Here, the control of RpoS stability was investigated in Salmonella typhimurium by the isolation of a number of mutants specifically defective in RpoS turnover. These included 20 mutants defective in mviA, the ortholog of Escherichia coli rssB/sprE, and 13 mutants defective in either clpP or clpX which encode the protease active on RpoS. An hns mutant was also defective in RpoS turnover, thus confirming that S. typhimurium and E. coli have identical genetic requirements for this process. Some current models predict the existence of a kinase to phosphorylate the response regulator MviA, but no mutants affecting a kinase were recovered. An mviA mutant carrying the D58N substitution altering the predicted phosphorylation site is substantially defective, suggesting that phosphorylation of MviA on D58 is important for its function. No evidence was obtained to support models in which acetyl phosphate or the PTS system contributes to MviA phosphorylation. However, we did find a significant (fivefold) elevation of RpoS during exponential growth on acetate as the carbon and energy source. This behavior is due to growth rate-dependent regulation which increases RpoS synthesis at slower growth rates. Growth rate regulation operates at the level of RpoS synthesis and is mainly posttranscriptional but, surprisingly, is independent of hfq function. (+info)
Natural synthesis of a DNA-binding protein from the C-terminal domain of DNA gyrase A in Borrelia burgdorferi.
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We have identified a 34 kDa DNA-binding protein with an HU-like activity in the Lyme disease spirochete Borrelia burgdorferi. The 34 kDa protein is translated from an abundant transcript initiated within the gene encoding the A subunit of DNA gyrase. Translation of the 34 kDa protein starts at residue 499 of GyrA and proceeds in the same reading frame as full-length GyrA, resulting in an N-terminal-truncated protein. The 34 kDa GyrA C-terminal domain, although not homologous, substitutes for HU in the formation of the Type 1 complex in Mu transposition, and complements an HU-deficient strain of Escherichia coli. This is the first example of constitutive expression of two gene products in the same open reading frame from a single gene in a prokaryotic cellular system. (+info)