Abstract：
:In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate faster through sucrose gradients (Noll, 2008). In fact, polysomes containing various numbers of ribosomes can be resolved from one another since each ribosome adds substantial mass. It is straightforward to prepare bacterial lysates and resolve all the ribosomal fractions using sucrose gradient sedimentation. The resulting polysome 'profile' can provide a sort of snapshot of the translation activity in the cell. Polysome analysis has been used to study the effects of mutations and/or growth conditions on translation and to address whether particular cellular components are associated with the translational machinery (Powers and Noller, 1990; Gregory et al., 1994; Moine and Dahlberg, 1994; Firpo et al., 1996; Fredrick et al., 2000; Ataide et al., 2009; Melamed et al., 2009; Saini et al., 2009). In combination with other techniques, polysome analysis has been used to deduce rate constants for certain phases of translation (e.g., initiation, elongation, termination) (Arava et al., 2003; 2005). Finally, use of purified polysomes in biochemical experiments has been instrumental for the isolation and characterization of translation factors such as ribosome recycling factor (RRF) (Hirashima and Kaji, 1972; Fujiwara et al., 2001; Hirokawa et al., 2002; Ito et al., 2002). Here, we describe a simple and convenient method of preparing and analyzing polysomes from Escherichia coli, which should be generally applicable to many bacteria. We also discuss parameters that influence the ribosome density on mRNA, which should be kept in mind when polysome profiles are being interpreted.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Qin D,Fredrick Kdoi
10.1016/B978-0-12-420037-1.00008-7subject
Has Abstractpub_date
2013-01-01 00:00:00pages
159-72eissn
0076-6879issn
1557-7988pii
B978-0-12-420037-1.00008-7journal_volume
530pub_type
杂志文章abstract：:It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2016.09.080
更新日期：2017-01-01 00:00:00
abstract：:Essentially the same steps are required to solve the crystal structure of a serpin as for any other protein: produce and purify protein, grow crystals, collect diffraction data, find estimates of the phase angles, and then refine and validate the structure. For the phasing step, experimental phasing methods involving ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385950-1.00004-3
更新日期：2011-01-01 00:00:00
abstract：:Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media for...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.08.031
更新日期：2015-01-01 00:00:00
abstract：:The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are formed by different Streptomyces strains and are potent inhibitors of bacterial gyrase. Their biosynthetic gene clusters have been analyzed in detail by genetic and biochemical investigations. Heterologous expression of these gene clusters ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(09)04618-7
更新日期：2009-01-01 00:00:00
abstract：:The target-of-rapamycin (TOR) pathway is a major regulator of cellular growth, division, and metabolism by coupling growth and nutrient cues to fundamental processes such as translation and transcription. In vertebrate models, disruptions affecting components of the TOR pathway result in cilia size abnormalities and c...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-397944-5.00009-2
更新日期：2013-01-01 00:00:00
abstract：:Considering that HIV-1 accumulates and replicates actively within lymphoid tissues, any strategy that will decrease viral stores in these tissues might be beneficial to the infected host. Follicular dendritic cells (FDC), B lymphocytes, antigen-presenting cells like macrophages, and activated CD4(+) T cells are abunda...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)91019-7
更新日期：2005-01-01 00:00:00
abstract：:Cellular signaling and regulatory cascades often rely on post-translational modification of proteins, particularly phosphorylation, to quickly and effectively relay signals from a variety of inputs. Numerous kinases, the effectors of phosphorylation, and kinase networks have been implicated in human diseases. Until re...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.07.017
更新日期：2019-01-01 00:00:00
abstract：:Respiration is widely used for evaluation of the metabolic capabilities or physiological state of the microbial culture. This chapter describes novel approaches for characterization of respiration at a single cell level: (1) flow cytometry-based redox sensing (FCRS) of actively metabolizing microbes; (2) respiration r...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-386905-0.00010-3
更新日期：2011-01-01 00:00:00
abstract：:Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-420067-8.00008-8
更新日期：2013-01-01 00:00:00
abstract：:Tumor cells acquire distinct genetic characteristics as a means to survive and proliferate indefinitely. Changes in the genetic code can also translate in changes at the protein level, therefore creating a distinguishable signature unique for tumor cells, and absent in normal tissues. The presence of discernable moiet...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.10.003
更新日期：2020-01-01 00:00:00
abstract：:Genetic modifier screens offer a powerful, indeed a uniquely powerful tool for the analysis and identification of elements capable of modulating specific cellular functions in development. Here, we describe the methodology that allowed us to explore the genetic circuitry that affects a Notch mutant phenotype caused by...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-397926-1.00016-0
更新日期：2014-01-01 00:00:00
abstract：:Human solid tumors are invariably less well-oxygenated than the normal tissues from which they arose. This so-called tumor hypoxia leads to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In this chapter, we examine the resistance of tumors to radiotherapy...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(07)35015-5
更新日期：2007-01-01 00:00:00
abstract：:Cryptococcus neoformans is a yeastlike fungus that causes a lethal meningoencephalitis in a broad spectrum of immunocompromised patients and has become the most common cause of meningitis due to AIDS-related infections in Africa. Key to the development of new agents to control and prevent this infection is the identif...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(08)03222-9
更新日期：2008-01-01 00:00:00
abstract：:For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting ...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/S0076-6879(07)31004-5
更新日期：2007-01-01 00:00:00
abstract：:Classifications and characterizations of specific proteins, such as enzymes, not only allow us to understand biosynthetic and metabolic pathways but they also help to drive our understanding of protein structure and function. How those characterizations are evaluated, however, may change our interpretations and lead u...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2017.06.024
更新日期：2017-01-01 00:00:00
abstract：:The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-405881-1.00005-7
更新日期：2013-01-01 00:00:00
abstract：:Differential scanning calorimetry (DSC) detects protein thermal unfolding by directly measuring the heat absorbed. Simple DSC experiments that require relatively small amounts of pure material can provide a wealth of information related to structure, especially with respect to domain architecture, without the need for...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.08.014
更新日期：2016-01-01 00:00:00
abstract：:Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrome...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2016.01.007
更新日期：2016-01-01 00:00:00
abstract：:The identification of novel forms of O-linked glycosylation on epidermal growth factor and thrombospondin type 1 repeats, and their emerging functional significance, require the development of new methods for their analysis. This chapter describes detailed methods to analyze both the structure and the site of modifica...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/S0076-6879(06)17008-1
更新日期：2006-01-01 00:00:00
abstract：:In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)09019-1
更新日期：2006-01-01 00:00:00
abstract：:As new organisms are being sequenced on a daily basis, new DNA glycosylases that recognize DNA damage can be easily identified in an effort to understand both their phylogenetics and substrate specificities. As a practical matter, existing bacterial and human homologs need to be readily available as laboratory reagent...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(06)08002-5
更新日期：2006-01-01 00:00:00
abstract：:Glycosylphosphatidylinositol-anchored proteins can be specifically identified by several methods. PI-PLC digestion analyses, the most widely used technique, can be performed more reliably when conducted with purified protein and phase partitioning to exclude steric effects and when combined with alkaline hydrolysis to...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/0076-6879(95)50099-5
更新日期：1995-01-01 00:00:00
abstract：:Coat protein complex II (COPII) vesicle-mediated protein export from the endoplasmic reticulum (ER) can be controlled by linear, independent motifs embedded within the cargo. ER export motifs directly interact with selective components of COPII vesicles and enhance cargo recruitment onto COPII vesicles. Moreover, ER e...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-391862-8.00010-7
更新日期：2013-01-01 00:00:00
abstract：:Proteins change their charge state through protonation and redox reactions as well as through binding charged ligands. The free energy of these reactions is dominated by solvation and electrostatic energies and modulated by protein conformational relaxation in response to the ionization state changes. Although computa...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/bs.mie.2016.05.052
更新日期：2016-01-01 00:00:00
abstract：:The methods described here concern the use specialized glassware to achieve accurate sub-micromolar to low-millimolar dioxygen concentrations specifically for the study of flavoproteins. Since the early 1960s, these methods have been passed through the lineage of flavin enzymologists almost exclusively as an oral trad...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.03.005
更新日期：2019-01-01 00:00:00
abstract：:The Microsealed Drug Delivery (MDD) system was developed to achieve several objectives: to maximize the bioavailability of a therapeutic agent in a target tissue; to minimize the adverse side effects of a therapeutic agent or its metabolites; to optimize the onset, rate, and duration of drug delivery; to maintain the...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/s0076-6879(85)12036-7
更新日期：1985-01-01 00:00:00
abstract：:Circadian rhythms generated by cell autonomous biological clocks allow for the appropriate temporal synchronization of physiology and behavior, optimizing the efficiency of biological systems. Circadian oscillators and functions have been uncovered in both central and peripheral tissues. This article describes methodo...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)93027-9
更新日期：2005-01-01 00:00:00
abstract：:An optimal tool to unravel the role of a specific player within a cellular network or process requires its spatiotemporally resolved perturbation. Chemically induced dimerization (CID) by the rapamycin system has proven useful to induce protein dimerization or translocation with high spatiotemporal precision. Recently...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2016.10.035
更新日期：2017-01-01 00:00:00
abstract：:Although the use of monoclonal antibodies to characterize the Ia antigen system is still in its infancy, the usefulness of this approach is clearly apparent. Since xenogeneic immunization can be used to generate these antibodies, the chances are good that a greater variety of Ia types and determinants will be detected...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/0076-6879(83)92010-4
更新日期：1983-01-01 00:00:00
abstract：:The three major considerations in reducing metal-gel binding are (1) the type of gel to be used, (2) the ionic strength of the eluent, and (3) alkaline reduction of the gel. Each of these parameters should be considered in advance of an experiment. A method is offered for determining the optimal gel type, gel treatmen...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/0076-6879(88)58044-8
更新日期：1988-01-01 00:00:00
