Probing the physical basis for trp repressor-operator recognition

Grillo, Adeola O.; Brown, Martha P.; Royer, Catherine A.

doi:10.1006/jmbi.1999.2625

Cited by 40 publications

(31 citation statements)

References 44 publications

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“…The value of FP is measured as anisotropy, which is a dimensionless parameter related to the intensity of parallel and perpendicular absorbed light components compared with the total intensity (31). The FP technique has been applied successfully to other DNA-binding protein systems, including CRP (32) and the tryptophan repressor (33). The following conditions were arrived at after testing effects of ionic strength, cation concentration, and protein and probe concentration (data not shown).…”

Section: IIImentioning

confidence: 99%

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

Thorsteinsson¹,

Kerby²,

Conrad³

et al. 2000

Journal of Biological Chemistry

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CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro 2 of one subunit (Met 1 is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the re- The sensing of dissolved gas molecules by proteins in biology has recently attracted considerable biochemical interest. The role of nitric oxide in a variety of important biochemical processes (1, 2), and its receptor, soluble guanylyl cyclase (sGC) 1 have been well documented in eukaryotic systems (3, 4). FixL, which modulates the expression of genes responsible for nitrogen fixation in rhizobia, is an example of an oxygen sensor (5, 6). An oxygen sensor in Escherichia coli, termed DOS ("direct oxygen sensor"), has been reported although its physiological role remains undefined (7). Finally, for carbon monoxide (CO), CooA, the CO-oxidation activator protein, modulates the expression of genes required for the utilization of CO as a sole energy source in the photosynthetic bacterium Rhodospirillum rubrum (8). All of the above mentioned proteins have in common a heme prosthetic group to which their respective gas molecules bind. The binding event is then followed by a conformational change in the protein that effects activity.Numerous studies have clearly demonstrated the physiological importance of CO in a wide variety of processes (9 -11), and although sGC has been implicated in sensing CO (12-14), direct evidence of a CO-receptor in eukaryotic signal transduction systems is lacking. CooA senses CO through a heme moiety and represents the current model system for biological CO-sensing (19,20). Finally, the ligand that is displaced upon binding CO remains speculative.Recently, the three-dimensional structure of Fe II CooA has been solved by x-ray diffraction techniques (21). This report showed that the general folding topology of CooA was indeed similar to that of CRP (22). In addition to the verification of His 77 as one of the heme-axial ligands in Fe II CooA, inspection of the structure identified the other axial ligand as an Nterminal proline residue (Pro 2 ; Met 1 is removed by processing) from the other subunit of the dimer. This structural environment represents an unprecedented axial ligation arrangement for a heme protein.In a previous study (18), we altered His 77 and found that the UV-visual spectra of these variants was normal in the Fe ʈ To whom correspondence should be addressed. Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts@bact.wisc.edu.1 The abbreviations used are: sGC, soluble guanylyl cyclase; CO, carbon monoxide; CRP, cAMP receptor protein; FixL, oxygen sensor of Rhizobium meliloti; Mb, myoglobin; P-4...

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Section: IIImentioning

confidence: 99%

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

Thorsteinsson¹,

Kerby²,

Conrad³

et al. 2000

Journal of Biological Chemistry

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“…This assay makes possible the separation of binding and catalytic parameters and the study of real-time kinetics. It is based on steady-state fluorescence anisotropy (r), which is sensitive to rotational diffusion, and thus suitable for studies aiming to identify structural modifications leading to a significant change in molecular size (12)(13)(14)(15). Using a fluorescent probe covalently linked to the GT dinucleotide, this makes it possible to follow DNA binding and dinucleotide release, because both steps strongly influence molecular size of the fluorescent moiety.…”

mentioning

confidence: 99%

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

Guiot

Carayon

Delelis

et al. 2006

Journal of Biological Chemistry

137

143

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The 3-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in realtime. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN⅐DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3-processing, with a dimer of dimers responsible for the subsequent full integration.The integration of a DNA copy of the HIV-1 2 genome into the host genome is a crucial step in the life cycle of the retrovirus. Integrase (IN) is responsible for the two consecutive reactions that constitute the overall integration process. The first of these two reactions is 3Ј-processing, which involves cleavage of the 3Ј-terminal GT dinucleotide at each extremity of the viral DNA. The hydroxyl groups of newly recessed 3Ј-ends are then used in the second reaction, strand transfer, for the covalent joining of viral and target DNAs, resulting in full-site integration. IN is sufficient for catalysis of the 3Ј-processing reaction in vitro, using short-length oligodeoxynucleotides (ODNs) that mimic one viral long terminal repeat (LTR) in the presence of the metallic cofactor Mg 2ϩ . This reaction generates two products: the viral DNA containing the recessed extremity and the GT dinucleotide. One of the two products, the processed viral DNA, as well as the target DNA serve as substrates for the subsequent joining reaction.IN belongs to the superfamily of polynucleotidyl transferases. Its catalytic core domain contains a triad of acidic residues constituting the D,D-35-E motif, which is strictly required for catalysis. The catalytic core establishes specific contacts with the viral DNA and, together with the C-terminal domain, is involved in DNA binding (1-4). ...

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“…The above described mechanism receives the name of repression, and is one of the three regulatory processes found in the trp operon. The binding of active repressors to the first two operators occurs cooperatively and this increases the sigmoidicity of the regulatory function accounting for repression [4,14]. Furthermore, it has been shown that a high level on sigmoidicity is necessary for the the bacteria to maintain a constant intracellular tryptophan concentration, regardless of variations in the amino acid concentration in the extracellular medium [16].…”

Section: Discussionmentioning

confidence: 99%

On the Use of the Hill Functions in Mathematical Models of Gene Regulatory Networks

Santillán

2008

Math. Model. Nat. Phenom.

144

108

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Abstract. Hill functions follow from the equilibrium state of the reaction in which n ligands simultaneously bind a single receptor. This result if often employed to interpret the Hill coefficient as the number of ligand binding sites in all kinds of reaction schemes. Here, we study the equilibrium states of the reactions in which n ligand bind a receptor sequentially, both non-cooperatively and in a cooperative fashion. The main outcomes of such analysis are that: n is not a good estimate, but only an upper bound, for the Hill coefficient; while the Hill coefficient depends quite strongly on the cooperativity level among ligands. We finally use these results to discuss the feasibility and constrains of using Hill functions to model the regulatory functions in mathematical models of gene regulatory networks.

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Probing the physical basis for trp repressor-operator recognition

Cited by 40 publications

References 44 publications

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

On the Use of the Hill Functions in Mathematical Models of Gene Regulatory Networks

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