PubMed Health⌕ Search

Biomedical subjects

M C Mossing

Publications and source records attributed to M C Mossing.

6 recordsLinked to original sources
PubMed1990

Stable, monomeric variants of lambda Cro obtained by insertion of a designed beta-hairpin sequence.

lambda Cro is a dimeric DNA binding protein. Random mutagenesis and a selection for Cro activity have been used to identify the contacts between Cro subunits that are crucial for maintenance of a stably folded structure. To obtain equivalent contacts in a monomeric system, a Cro variant was designed and constructed in which the antiparallel beta-ribbon that forms the dimer interface was replaced by a beta-hairpin. The engineered monomer has a folded structure similar to wild type, is significantly more stable than wild type, and exhibits novel half-operator binding activity.

Amino Acid Sequence↗
PubMed1988

Physical properties of DNA in vivo as probed by the length dependence of the lac operator looping process.

Plasmid constructs containing a wild-type (O+) lac operator upstream of an operator-constitutive (Oc) lac control element exhibit a length-dependent, oscillatory pattern of repression of expression of the regulated gene as interoperator spacing is varied from 115 to 177 base pairs (bp). Both the length dependence and the periodicity of repression are consistent with a thermodynamic model involving a stable looped complex in which bidentate lac repressor interacts simultaneously with both O+ and Oc operators. The oscillatory pattern of repression with distance occurs with a period approximating the helical repeat of DNA and presumably reflects the necessity for proper alignment of interacting operators along the helical face of the DNA. In the length regime examined, the presence of the upstream operator enhances repression between 6-fold and 50-fold depending upon phasing. This reflects a torsional rigidity of DNA in vivo that is consistent with in vitro measurements. The oscillatory pattern of repression is best fit with a period of either 9.0 or 11.7 bp/cycle but not 10.5 bp/cycle. This periodicity is interpreted as reflecting the average helical repeat of the 40-bp interoperator region of plasmid DNA in vivo, suggesting that the local helical repeat of DNA in vivo may differ significantly from 10.5 bp/turn. The apparent persistence length needed to fit the data (aapp) is only one-fifth the standard in vitro value. This low value of aapp may be due in part to DNA bending induced by catabolite activator protein (CAP) bound to its site between the interacting operators.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗
PubMed1987

Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression.

Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.

Amino Acids↗
PubMed1986

Upstream operators enhance repression of the lac promoter.

To study regulation of transcription by distant elements, a wild-type lac operator was inserted upstream of a promoter-constitutive operator control region. The upstream operator is shown to aid in repression of transcription from the mutant control region. The effectiveness of the upstream operator as a function of its distance from the mutant control region parallels the length dependence observed for DNA cyclization. A quantitative model is proposed for action-at-a-distance of DNA control sites in which protein-protein and protein-DNA interactions are mediated by DNA looping. In this model, the effective concentrations of interacting proteins that are tethered by DNA are determined by the length of the intervening DNA and by its inherent bending and torsional stiffness. This model makes a number of predictions for both eukaryotic and prokaryotic control sequences located far from their sites of action.

Galactokinase↗