scholarly journals The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer

2004 ◽  
Vol 186 (1) ◽  
pp. 1-7 ◽  
Author(s):  
David R. Pawlowski ◽  
Gerald B. Koudelka
Keyword(s):  
Dna Binding ◽  
Transcriptional Regulatory ◽  
Dna Binding Site ◽  
Cleavage Step ◽  
Regulatory Functions ◽  
Bacteriophage 434 ◽  
Dna Complex ◽  
Bacteriophage 434 Repressor ◽  
434 Repressor

ABSTRACT Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein. Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA. Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo. Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA. Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer. DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers. These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage. We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA. Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step. Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor.

Synthesis and mass spectrometric characterisation of the N-terminal (1-63) DNA-binding domain of the bacteriophage 434 repressor cI

1997 ◽  
Vol 3 (1) ◽  
pp. 151
Author(s):  
Piergiorgio Percipalle ◽  
Sandor Pongor ◽  
Sotir Zakhariev ◽  
Corrado Guarnaccia ◽  
Rosaria Saletti ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mass Spectrometric ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Bacteriophage 434 ◽  
Bacteriophage 434 Repressor ◽  
434 Repressor

scholarly journals Functional Domains of the TOL Plasmid Transcription Factor XylS

2000 ◽  
Vol 182 (4) ◽  
pp. 1118-1126 ◽  
Author(s):  
Niilo Kaldalu ◽  
Urve Toots ◽  
Victor de Lorenzo ◽  
Mart Ustav
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dependent Manner ◽  
Tol Plasmid ◽  
Terminal Fragment ◽  
Central Regions ◽  
Basic Features ◽  
Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

scholarly journals The Bacteriophage 434 Repressor Dimer Preferentially Undergoes Autoproteolysis by an Intramolecular Mechanism

2005 ◽  
Vol 187 (16) ◽  
pp. 5624-5630 ◽  
Author(s):  
Barbara C. McCabe ◽  
David R. Pawlowski ◽  
Gerald B. Koudelka
Keyword(s):  
Dna Damage ◽  
Active Sites ◽  
Sos Response ◽  
Lambdoid Phage ◽  
Lambdoid Prophage ◽  
Operator Binding ◽  
Bacteriophage 434 ◽  
Bacteriophage 434 Repressor ◽  
434 Repressor ◽  
Intramolecular Mechanism

ABSTRACT Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.

scholarly journals Genetic Analysis, Using P22 Challenge Phage, of the Nitrogen Activator Protein DNA-Binding Site in the Klebsiella aerogenes put Operon

1998 ◽  
Vol 180 (3) ◽  
pp. 571-577 ◽  
Author(s):  
Li-Mei Chen ◽  
Thomas J. Goss ◽  
Robert A. Bender ◽  
Simon Swift ◽  
Stanley Maloy
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Genetic Analysis ◽  
Nitrogen Assimilation ◽  
Regulatory Protein ◽  
Klebsiella Aerogenes ◽  
Dna Binding Site ◽  
Control Protein

ABSTRACT The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons fromKlebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

scholarly journals A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

2021 ◽  
Author(s):  
Shalini Gupta ◽  
Larry J. Friedman ◽  
Jeff Gelles ◽  
Stephen P. Bell
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Single Molecule ◽  
Rapid Change ◽  
Single Molecule Spectroscopy ◽  
Replication Origins ◽  
Helicase Loading ◽  
Dna Binding Site ◽  
Opposite Face ◽  
Dna Complex

AbstractReplication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

scholarly journals Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 149 ◽  
Author(s):  
Monika Karney ◽  
Joy McKenna ◽  
Natasha Weatherspoon-Griffin ◽  
Alexander Karabachev ◽  
Makensie Millar ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Inverted Repeat ◽  
Virulence Genes ◽  
In Vivo Studies ◽  
Single Nucleotide ◽  
Binding Interactions ◽  
Shigella Species ◽  
Dna Binding Site

The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed.

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