遗传学实验技术

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 ...

Trans-translation is a bacterial quality control system in protein synthesis facilitated by transfer-messenger RNA (tmRNA). Here, we describe the in vitro system using purified factors to evaluate the two steps of trans-translation: peptidyl-transfer from peptidyl-tRNA to ala ...

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences composed of a succession of repeats (23–50 bp long) separated by unique sequences called spacers. CRISPRs together with a set of genes called cas for CRISPR associated, constitute a defence mechan ...

Diverse bacteria encode RNAs that are not translated into proteins but are instead involved in regulating a wide variety of cellular functions. Computational approaches have proven successful in identifying numerous regulatory RNAs in myriad bacterial species but the difficul ...

Whole-genome tiling arrays are powerful tools for detecting and characterizing novel RNA transcripts. Here, we describe a complete method combining elements of molecular and computational biology to identify small noncoding RNA (sRNA) transcripts. We focus on the key features of th ...

Small regulatory RNAs (sRNAs) are versatile regulators that have been shown to be involved in the gene regulation of a growing number of biological pathways in bacteria. While finding the targets of a given sRNA has been the focus of many studies, fewer methods have been described to uncover which, if a ...

The Northern blot technique is widely used to study RNA. This relatively old method allows one to detect RNA molecules ranging in size from ∼20 to thousands of nucleotides and simultaneously estimate the size of an RNA and detect its degradation/processing products. The method does not rely on en ...

Ordered collections of barcoded deletion mutants can be screened rapidly in mixed cultures to uncover resistance and sensitivity phenotypes associated with the loss of a gene. As such, they are invaluable tools for assigning gene function in the post-genomic era. In this protocol, we descr ...

Milligram quantities of RNA are commonly synthesized by in vitro transcription from a DNA template with T7 RNA polymerase. However, the run-off transcription method results in heterogeneity at the RNA 3′-terminus. RNA purification requires single-nucleotide resolution to separ ...

During in vitro run-off transcription with T7 RNA polymerase, transcripts with heterogenous 3′ ends are commonly synthesized. Here, we describe an efficient procedure for correct processing of transcript 3′ ends with the use of antigenomic HDV ribozyme. The procedure involves the exte ...