Time-resolved emission spectroscopy as a tool to follow nucleic acid-protein interaction.

Fluorescence spectroscopy is undoubtedly a useful tool to study the structural and functional aspects of nucleic acids-protein interactions as well as the catalytic functions of particular residues of multi-subunit enzyme complexes. The dynamic interaction of nucleic acids and proteins occurring at nanosecond time scale can now be monitored by making life-time measurements or by time-resolved emission spectroscopy. These measurements are made by exploiting the intrinsic fluorescent residues in proteins i.e. W or by the use of extrinsic fluorophores which are tagged on to particular residues and that are sensitive to the microenvironment changes. In this study we describe the use of time resolved emission spectroscopy to (a) analyse the transient binding between sigma 70 and DNA by monitoring the quenching of W residues and (b) monitor the various states which nucleosomes of active, inducible or inactive chromatin may adopt in vivo.

Molecular anatomy of the transcription complex of Escherichia coli during initiation.

Transcription is the foremost event in gene expression in which the enzyme RNA polymerase copies the genetic information from DNA to RNA. Much of our understanding of this process have come from studies carried out in Escherichia coli. A faithful and efficient transcription machinery of E. coli can be reconstituted in vitro with purified RNA polymerase and promoter-containing DNA. It is generally believed that in E. coli and most other organisms, the control of gene expression lies with the initiation of transcription. In this review, an attempt has been made to understand the mechanistic details of the initiation of transcription from the structural point of view of the promoter and the RNA polymerase. Allosteric nature of the enzyme has also been discussed at the end.

Conformational and ion-binding properties of cyclolinopeptide A isolated from linseed.

The conformation of the cyclic nonapeptide from linseed, cyclolinopeptide A in methanol and in acetonitrile has been elucidated by one- and two-dimensional nuclear magnetic resonance. The molecule is folded in a ß-turn conformation. Cyclolinopeptide A interacts and weakly complexes with Tb3+ (a Ca2+ mimic ion) with the metal ion positioned proximally to the Phe residue, but with no substantial structural alteration upon metal binding. Cyclolinopeptide A is also seen to aid the translocation of Pr3+ (another Ca2+ mimic) across unilamellar liposomes. However, cyclolinopeptide A does not phase transfer or act as an ionophore of calcium ion myself. Experiments using lanthanide ions thus do not necessarily indicate any ionophoretic ability of the complexone towards calcium ions.

Spectroscopic studies on the denaturation of papain solubilized and Triton X-100-solubilized glucoamylase from rabbit small intestine.

Intestinal brush border proteins consist of an enzymatically active hydrophilic moiety attached to a hydrophobic tail. Papain dissociates the hydrophilic part by cleaving off the hydrophobic tail, whereas the detergentTriton X-100 solubilizes the whole molecule. Denaturation by 8 Μ urea or 4 Μ guanidinium chloride does not alter the structure of the papain-solubilized enzyme. An appreciable alteration of the structure of detergent-solubilized enzyme was observed on denaturation. The difference spectra of Triton X-100 (1%)—solubilized enzyme and its urea denatured form shifts and intensifies, with increase in the concentration of the denaturant with an isobestic point at 252 nm. A new band at 280 nm also appears at 4 Μ urea concentration. Papain-solubilized glucoamylase has an ∝ -helical conformation in solution unlike the detergentsolubilized fraction. An elongated structure for the papain solubilized enzyme is inferred from the urea denaturation studies and from molecular weight determinations.