We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.

The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.

Successful development of plants resistant to salinity stress is problematic as a result of the complex polygenic natures of salt tolerance. Previously, alkaline extracts of the brown seaweed Ascophyllum nodosum have shown promise in enhancing plant tolerance toward abiotic stresses. To understand the underlying molecular mechanisms, the whole genome transcriptome of Arabidopsis undergoing salt stress was analyzed by microarray analysis after treatment with the chemical components of A. nodosum extracts (ANE). Treatment with ANE induced many positive regulators of salt tolerance in addition to downregulating numerous other genes. Using T-DNA insertion mutants within these downregulated genes, we examined the potential for a novel source of enhanced NaCl tolerance through removal of negative regulators of NaCl stress responses within Arabidopsis. Several potential target mutations were identified with enhanced salt-tolerant phenotypes. A T-DNA insertion within the promoter of a putative Pectin Methyl Esterase Inhibitor (PMEI) gene (At1g62760) was found to be resistant to salinity stress and was further characterized. This T-DNA insertion mutant was designated as pmei1-1. The phenotype of pmei1-1 seedlings included increased primary root growth in vitro and improved biomass accumulation under NaCl stress. Additionally, modified transcript levels of dehydration-responsive genes, including RD29A, were observed in pmei1-1 plants. Taken together, these results suggest a role for PMEI as a negative regulator of NaCl resistance and that chemical stress-induced transcriptome analysis may lead to identification of additional novel regulators of abiotic stress tolerance in plants, the use of which would have significant implications for agriculture globally.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites. Each met repressor dimer is centered on a met box and interacts with the adjacent dimer through antiparallel alpha-helices, which explained the observed cooperative nature of the binding. DNA binding takes place through the insertion of a beta-ribbon into the major groove of B-form DNA, representing a novel DNA binding motif. Sequence specificity arises from direct interactions between side chains of the beta-strands and the edges of the bases in the major groove. The local DNA conformation confers additional specificity through interactions between protein and the phosphate backbone. The repressor is activated through binding of S-adenosyl methionine (SAM), the corepressor, to the face opposite to that used for DNA binding. The lack of significant conformational change upon SAM binding, together with electrostatic calculations, suggests that DNA binding enhancement occurs through long-range electrostatic interactions.