Real-Time Observation of Backtracking by Bacterial RNA Polymerase

RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA–RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking

Detection Methodology Based on Target Molecule-Induced Sequence-Specific Binding to a Single-Stranded Oligonucleotide

We have recently developed a mix-and-read format homogeneous antigen peptide based assay for detection of the antibodies (Tian, L.; Heyduk, T. <i>Anal. Chem.</i> <b>2009</b>, <i>81</i>, 5218–5225) that employed for target detection a simple biophysical mechanism of target antibody induced annealing between two complementary oligonucleotides attached to the antigen peptide. In this work, we propose and experimentally validate an alternative variant of this assay format in which target antibody binding to antigen peptide–oligonucleotide conjugate produces a complex with high sequence-specific binding affinity to a single-stranded capture oligonucleotide. This new assay format can be used for preparing various solid-surface based assays by immobilizing the capture oligonucleotide. This assay design is not limited to antibody detection. We demonstrate that it can also be employed for detecting proteins or pathogenic bacteria using oligonucleotide-labeled antibodies as target recognition elements. Preparation of these solid-surface based assays is simplified because all interactions with the solid surfaces are mediated by well-understood oligonucleotide–oligonucleotide interactions and because of the relative ease of immobilizing oligonucleotides on various solid surfaces. These unique aspects of the assay design also allow microarray-style multiplexing that could be most useful for multiplexed antibody profiling for diagnosis and analysis of cancer, autoimmune, and infectious diseases

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, β-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI &lt; 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains

RNA polymerase motions during promoter melting.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety

A total of 31 of thiourea derivatives was prepared reacting 3-(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yields varied from 35% to 82%. All compounds were evaluated in vitro for antimicrobial activity. Derivatives 3, 5, 6, 9, 15, 24 and 27 showed the highest inhibition against Gram-positive cocci (standard and hospital strains). The observed MIC values were in the range of 0.25e16 μg/ml. Inhibitory activity of thioureas 5 and 15 against topoisomerase IV isolated from Staphylococcus aureus was studied. Products 5 and 15 effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Moreover, all obtained thioureas were evaluated for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses. Compounds 5, 6, 8e12, 15 resulted cytotoxic against MT-4 cells (CC50≤ 10μM)