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Analysis of physiological mechanisms that control transcription often requires extrapolation of in vitro measurements into in vivo mechanisms. This extrapolation is complex, as mammalian genes are commonly organized into broad chromosomal domains, and such domains cannot be readily reconstituted in vitro. Thus, the nucleoprotein structure of chromosomes constitutes a considerable impediment to elucidating...
In their article, A.S. Weinmann and P.J. Farnham (2002, Methods 26) described new techniques for isolating in vivo binding sites for any DNA-binding protein. In this article, we describe complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions. First, we describe how formaldehyde crosslinking and chromatin immunoprecipitation (ChIP), in conjunction with...
With the increasing popularity of the yeast two-hybrid screen, a large number of protein-protein interactions have been identified. In many cases, single proteins have been found to associate with a large number of cofactors. For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2...
Critical to understanding biological roles for transcription factors is an appreciation of the target genes regulated by the transcription factor. The identification of target genes can often expand the understanding of known biological roles for a transcription factor and may reveal unappreciated and unexpected functions. This article focuses on the identification and characterization of transcription...
In vivo footprinting techniques are useful for the identification of regulatory elements mediating transcriptional control of a gene. However, regulation of a gene can differ between the two alleles, and further steps must be taken to distinguish between the regulatory elements occupied on one allele and those used on the second allele. Many hematologic malignancies result from chromosomal translocations,...
This article contains a detailed protocol for localizing transcription factors on Drosophila melanogaster polytene chromosomes by immunofluorescence. The large polytene chromosomes from third-instar larval salivary gland cells allow mapping of chromosome-associated proteins at high resolution. Thus, this method has been used to investigate how broadly transcription factors function and to identify...
The standard chromatin immunoprecipitation (ChIP) assay is used to examine the specific association of transcription factors with DNA in the context of living cells. Here we review two modifications to this protocol which are designed to identify novel target genes of transcription factors in mammalian cells. The main advantage to both of these approaches is that only DNA sequences directly bound...
The once ambitious goal of creating custom DNA-binding factors has been achieved. Advances in construction methodology now enable any laboratory to create site-specific binding proteins to nearly any sequence. Using predefined zinc finger modules, new proteins can be constructed in days with minimal cost and using only standard polymerase chain reaction techniques. The existing spectrum of modules...
Recent data support the idea that the mammalian nucleus is organized in a functionally significant way. Immunocytochemistry has revealed the existence of diverse nuclear ''bodies'' and compartments. Fluorescence in situ hybridization (FISH) has shown that chromosomes change their spatial relationships during dynamic cell cycle progression and that nuclear organization can change during development...
Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification...
The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent...
Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a ''functional'' domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from...
Much attention is currently being devoted to questions of protein and RNA tertiary structures and to the quaternary arrangement of the individual macromolecules in ribonucleoprotein (RNP) particles. In this article we describe two complementary strategies that allow the identification of RNA-protein contact sites in assembled, nonlabeled RNP particles after UV crosslinking. The first combines immunoprecipitation...
RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically,...
Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two...
Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for...
Messenger RNA transport has emerged as a significant mechanism for regulating gene expression. Many of the protein factors affecting RNA transport remain unknown. The emergence of green fluorescent protein (GFP) fluorescence microscopy allows imaging in living cells and an increased understanding of in vivo molecular transport. GFP imaging is now applied to RNA transport by engineering RNA hairpins...
Although in vitro methods have been used to identify putative targets of mRNA-binding proteins, direct in vivo methods are needed to identify endogenously associated mRNAs and their cognate proteins. Therefore, we have developed high-throughput methods to identify structurally and/or functionally related mRNA transcripts through their endogenous association with RNA-binding proteins. We have termed...
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