The interaction of DNA topology modifying enzymes with eukaryotic DNA replication origins can be detected with nucleotide precision exploiting the action of enzyme poisons specific for type I or type II DNA topoisomerases. Using the topoisomerase I poison camptothecin and the topoi ...

In vertebrate cells, DNA topoisomerase II (Topo II), named Top2 in yeast, localizes along chromosome axes early in mitosis and concentrates within centromeric chromatin during metaphase. The factors controlling these changes in enzyme distribution are largely unknown. Insight in ...

Here we describe an adapted ChIP-on-chip protocol for the analysis of DNA topoisomerase chromosomal binding in Saccharomyces cerevisiae cells. The ChIP-on-chip technique is based on the immunoprecipitation of crosslinked chromatin (ChIP, chromatin immunoprecipitation), ...

In eukaryotic cells, topoisomerase III forms an evolutionarily conserved complex with a RecQ family helicase and two OB-fold containing proteins, replication protein A (RPA) and RMI1. One role for this complex is to catalyze the completion of homologous recombination reactions in whi ...

The past few years have seen the application of single-molecule force spectroscopy techniques to the study of topoisomerases. Magnetic tweezers are particularly suited to the study of topoisomerases due to their unique ability to exert precise and straightforward control of the supe ...

Type II DNA topoisomerases are essential, ubiquitous enzymes responsible for performing vital functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) performs these DNA transactions by passing one ...

To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of li ...

Detecting localized RNA in bacteria is difficult due to the properties of RNA and the small size of the cell. Fluorescence in situ hybridization (FISH) has been an invaluable method for detecting and imaging RNA. In FISH, RNA is fixed in its native subcellular position through chemical cross-lin ...

Noncoding RNAs serve myriad functions in the cell, but their biophysical properties are not well understood. Calorimetry offers direct and label-free means for characterizing the ligand-binding and thermostability properties of these RNA. We apply two main types of calorimetry—i ...

RNA is now appreciated to serve numerous cellular roles, and understanding RNA structure is important for understanding a mechanism of action. This contribution discusses the methods available for predicting RNA structure. Secondary structure is the set of the canonical base pairs, a ...

A broad range of biological processes relies on complexes between RNA and proteins. Crystallization of RNA–protein complexes can yield invaluable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA–protein compl ...

Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our un ...

Endoribonuclease footprinting is an important technique for probing RNA–protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein bind ...

The gel mobility shift assay is a powerful technique for detecting and quantifying protein–RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein–RNA interactions, gel shift analysis p ...

Many small noncoding RNAs (sRNAs) in bacteria act as posttranscriptional regulators by base pairing to their message targets. TargetRNA is a program that predicts the targets of a sRNA by identifying messages with significant potential to base pair with the sRNA. Since base pairing potent ...

RNA–protein interactions are critical in diverse aspects of gene expression and often serve to mediate regulatory events. Many procedures are available to gain information about RNA–protein interactions. They span from initial identification of an interaction, such as through co ...

Northern blots are extremely useful to monitor the steady state level of small regulatory RNAs (sRNAs) as well as their target mRNAs. In combination with the drug rifampicin, which blocks cellular transcription, Northern blots can be used to determine the stability of sRNAs and mRNAs. Here we de ...

Electrophoretic mobility shift assay is a simple, rapid, and sensitive technique to analyze the RNA–RNA interaction. A 32P-labeled RNA is incubated with another unlabeled RNA and subjected to electrophoresis on a native polyacrylamide gel. If two RNA molecules base pair stably, the move ...

Ribosomes are large complexes of RNA and protein that perform the essential task of protein synthesis in the cell. Ribosomes also serve as the initiation point for several translation-associated functions. To perform these tasks efficiently, ribosomes interact with a myriad of nonrib ...

During protein synthesis, ribosomes translate the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent pep ...