The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...
The concentration of phenols, procyanidins, and protein in pine foliage was determined at two and eight weeks following mechanical defoliation of 0, 25, 50, and 75%. Changes in both mature and immature ponderosa pine, Pinus ponderosa Dougl. ex Laws, foliage were monitored. The lowest level of defoliation (25%) produced the largest increase in phenols; 75% defoliation caused the largest increase in procyanidins. The number of phenolic compounds in mature foliage doubled following 25% defoliation. Maximum protein production also occurred at moderate defaliation levels. Mechanical defoliation had a significant effect on the production of all compounds tested. The implications of these results for induced resistance research are discussed.
The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpR s , gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer±DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.
To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 'H-NMR spectroscopy. We report here the 'H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.Keywords: Trp repressor; deuteration ; trp operator.The Escherichia coli Trp repressor is activated by tryptophan to bind to at least five operators in the E. coli genome, namely the trpEDCBA (trpO), aroH, aroL, trpR and mtr operators (reviewed in [I]) [ 2 ] . It thereby represses initiation of transcription of genes involved in tryptophan uptake and biosynthesis in response to intracellular levels of tryptophan. As one of the smallest proteins whose binding to a specific DNA sequence is allosterically controlled, the Trp repressor has been studied extensively by a variety of genetic and physical methods; however, the basis for its DNA selectivity remains controversial.The structures of the protein and several of its complexes have been determined by X-ray crystallography [3-71 and, to lower resolution, by 'H and heteronuclear NMR spectroscopy [8-131. The protein belongs to the helix-turn-helix family of dimeric DNA-binding proteins. Tryptophan binds between the core of the molecule and the DNA-binding helices in each subunit, changing their orientation so that they are better positioned to bind to the DNA. In the crystal structure of the holoprotein bound to a double-stranded oligodeoxynucleotide containing a symmetrised version of the trpO operator sequence, the corepressor tryptophan also interacts directly with the phosphate backbone of the DNA [6]. Surprisingly in this structure there are no direct hydrogen bonds or van der Waals' interactions between the protein and the base pairs of the DNA known to be important for sequence-specific binding. There are several water-mediated interactions that could be important for specificity. Alternatively, specificity could arise from the interactions of the protein with the phosphate backbone ('indirect readout'). This result has been controversial: an NMR study of the same co...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.