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Efficient manipulation of the regulatory mechanisms controlling host cell gene expression provides the means for productive infection by animal viruses. Upon infecting the host cell, viruses must: (i) bypass the cellular antiviral defense mechanisms to prevent the translational blocks imposed by the interferon pathway; and (ii) effectively “hijack” the host protein synthetic machinery into mass production...
After a brief introduction to the function of the mRNA-specific translation factors eIF4A, eIF4B, and eIF4F, this article presents appropriate methodology for the study of the translation factors associated with the activation of mRNA for translation in eukaryotic systems. The purification of eIF4A, eIF4B, and eIF4F from rabbit reticulocyte lysates is given along with a procedure for the purification...
The biosynthesis of RNAin vitrousing bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays. PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation...
Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the α subunit of the translation...
The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the α-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2α phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts...
Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular...
Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitablein vitrotranslation system in which the mode of action of certain translation factors can be studied. This report describes the preparation...
Nucleosomes and the chromatin structures they assemble provide the architectural foundation for the transcription process. We describe the use of reconstituted chromatin templates assembled using purified histones and HMGs to investigate the significance of chromatin structure for transcription. Our structural assays include sucrose gradient fractionation, nucleoprotein gel analysis, micrococcal nuclease...
The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are...
A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of...
Electron microscopy, with its ability to image DNA and nucleosomes, can provide a key visual link in the understanding of chromatin conformation. We discuss applications of EM to current chromatin research with emphasis on strategies that eliminate many of the potential problems associated with conventional EM preparative techniques. Cryo-electron microscopy (cryo-EM) of isolated chromatin, whereby...
Our current level of understanding of chromatin structure was to a large extent achieved with the help of DNA–protein cross-linking. The versatile inventory of cross-linking techniques allows the identification of the contacts between DNA and proteins with a single nucleotide–single amino acid precision, to detect minor components of the complex nucleoprotein systems, to reveal the interactions of...
Nucleoprotein gel electrophoresis, as well as its use in chromatin research, is reviewed from its early application in the characterization of native nucleosome composition to current uses in analyzing transcription factor–nucleosome complexes and in visualizing multiple nucleosome positioning. Despite our incomplete understanding of the principles behind the separation of particles in native polyacrylamide...
Analytical ultracentrifugation and agarose gel electrophoresis each can be used to accurately quantify changes in structure that accompany chromatin folding in solution. Analytical ultracentrifugation directly measures the extent of compaction of each species present in a chromatin sample under a wide range of solution conditions. Agarose gel electrophoresis yields information about changes in the...
Several promoters have been shown to have sequence specifically positioned nucleosomes that determine the architecture of the promoter. DNA binding proteins that regulate gene expression are in many cases known to bind to their cognate DNA segments organized within such positioned nucleosomes. It has become increasingly evident that the cooperation of chromatin and transcription factors results in...
Acetylation of specific lysine residues in the N-terminal domains of core histones is a biochemical marker of active genes. To determine the spatial and temporal distribution of this reversible posttranslational modification, affinity-purified polyclonal antibodies recognizing the epitope ϵ-acetyllysine have been used in immunoselection procedures with mononucleosomes and salt-soluble chromatin fragments...
Increasingly, biochemical analyses of processes that occur within eukaryotic nuclei such as transcription and replication require the construction of specific chromatin substrates. Nucleosome complexes reconstitutedin vitrohave been key elements in a variety of recent studies of polymerase progression andtrans-acting factor binding activities. Reconstituted complexes can be easily constructed from...
A two-dimensional electrophoresis for fine separation of histones is described in detail. The method is relatively simple and gives very reproducible results. In the first dimension the histones are separated by their charge in acid–urea gels, while in the second dimension the separation is based on both the charge and the differential affinity of histones to Triton in acid–urea–Triton gels. In this...
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