GATC flanking sequences regulate Dam activity: evidence for how Dam specificity may influence pap expression

Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E.coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E.coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E.coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam.     

Protein-induced DNA bending clarifies the architectural organization of the sigma54-dependent glnAp2 promoter

Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.     

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.     

Cooperative binding of the leucine-responsive regulatory protein (Lrp) to DNA

The leucine-responsive regulatory protein (Lrp) of Escherichia coli activates expression of a number of operons and represses expression of others. For some members of the Lrp regulon, exogenous leucine mitigates the effect of Lrp, for some it potentiates the effect of Lrp, and for others it has no effect on Lrp action. For the ilvIH operon that we study, Lrp activates expression in vivo and mediates the repression of the operon by exogenous leucine. We studied Lrp-1, a leucine-insensitive variant, to investigate mechanisms by which leucine alters Lrp action as an activator of ilvIH expression. The Asp114Glu change did not have much effect on the amount of total Lrp-1 in cells but decreased the amount of free Lrp-1 two- to threefold. Lrp monomers associate to form octamers and hexadecamers (hexadecamer form predominates at micromolar concentrations; Kd=5.27x10(-8) M), and leucine promotes the dissociation of Lrp hexadecamer to a leucine-bound octamer. By contrast, Lrp-1 exists primarily as an octamer in solution (equilibrium dissociation constant 6.5x10(-5) M) and leucine had little effect on the equilibrium. Thus, the hexadecameric form that Lrp assumes in the absence of DNA is not required for activation of the ilvIH operon. Both leucine and the lrp-1 mutation reduced the apparent affinity of Lrp binding to ilvIH DNA (contains two groups of binding sites separated by 136 bp) but they have different effects on intrinsic binding affinity and binding cooperativity. Whereas leucine reduced intrinsic binding affinities and interactions of Lrps bound at upstream and downstream regions of ilvIH DNA, it increased cooperative dimer-dimer interactions of Lrps bound to two adjacent sites. By contrast, the lrp-1 mutation did not have much effect on intrinsic binding affinities but it decreased cooperative adjacent dimer-dimer interactions and enhanced interactions of Lrps bound at upstream and downstream regions of ilvIH DNA. Our analysis is consistent with the idea that leucine enhances dimer-dimer interactions that contribute to octamer formation, concomitantly reducing dimer-dimer interactions that contribute to the longer range interactions of Lrps that are required for activation of the ilvIH promoter.