Maxim Kozlov

Mapping of Catalytic Residues in the RNA Polymerase Active Center

When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the β′ subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutation. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex. The mutant fails to support Fe2+-induced cleavage of DNA or protein. Thus, the NADFDGD motif is involved in chelation of the active center Mg2+.

Donation of catalytic residues to RNA polymerase active center by transcription factor Gre

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3′ RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg2+ ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg2+ ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.

Luminescent probes for ultrasensitive detection of nucleic acids.

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Strategies and Methods of Cross-Linking of RNA Polymerase Active Center

Publisher Summary This chapter presents the basic strategies and methods of cross-linking of RNA polymerase (RNAP) active center. In order to achieve the high selectivity of affinity labeling for RNA polymerase, it is required to take advantage of “catalytic competence.” This phenomenon reflects the ability of a substrate residue cross-linked at the active center of an enzyme to convert to a cross-linked product by the same enzyme molecule according to the normal mechanism of catalysis. At the first stage RNAP is treated in the binary complex with a promoter by affinity reagent, which is an analog of initiating substrate. This results in the cross-linking of affinity reagent residues both inside and outside the active center. At the second stage the modified enzyme is supplemented with the second radioactive substrate complementary to the next base of DNA template. The chapter discusses RNA–protein cross-linking in the active center of initiating and elongation. Single-Hit degradation of polypeptides at particular residues is also discussed.. Multiple cross-linking sites can be revealed by quantitative analysis of single-hit degradation products. Another approach that appears to be very helpful for the mapping is based on the usage of functionally active enzymes assembled from the fragments of RNAP subunits.

New cross-linking quinoline and quinolone based luminescent lanthanide probes for sensitive labeling

New luminescent lanthanide chelates containing thiol-, amine-, and click-reactive groups in antenna-fluorophore moieties were synthesized. The chelates include diethylenetriaminepentaacetic acid (DTPA) coupled to two types of chromophores: 7-amino-4-trifluoromethyl-2(1H) quinolinone, and 7-amino-4-trifluoromethyl-2-alkoxyquinoline. The synthesized compounds were characterized using NMR, light absorption, steady-state and time-resolved fluorescent spectroscopy. Some of the compounds displayed high brightness with Tb3+, Eu3+, and Dy3+. Obtained reactive lanthanide chelates can be easily attached to biological molecules. The probes demonstrated high performance in molecular beaconbased DNA hybridization assays (sub-pico molar detection limit), in bacterial proteome labeling, and in live cell imaging.