RNA Turnover in Bacteria, Archaea and Organelles
Methods in Enzymology Series, Vol. 447

Coordinators: Maquat Lynne E., Arraiano Cecilia

Language: Anglais

Subject for RNA Turnover in Bacteria, Archaea and Organelles

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450 p. · 15.2x22.9 cm · Hardback
Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements, responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation.

Covers the difference in processing of mRNA between eukaryotes, bacteria and archea. Benefit: Processing of mRNA differs greatly between eukaryotes, bacteria and archea and this affords researchers readily reproducible techniques to understand and study the molecular pathogenesis of disease.

Expert researchers introduce the most advanced technologies and techniques to identify mRNA processing, transport, localization and turnover which are central to the process of gene expression. Benefit: Keeps MIE buyers and online subscribers up-to-date with the latest research

Offers step by step lab instructions including necessary equipment and reagents. Benefit: Provides tried and tested techniques which eliminate searching through many different sources. Tested techniques are trustworthy and avoid pitfalls so the same mistakes are not made over and over.
Part I Bacteria

Analysis of RNA decay, including polyadenylation, in mutant E. coli
Analyzing the decay of stable RNAs in E. coli
Genomic analyses of mRNA decay in E. coli using microarrays
Methods to co-immunopurify degradative activities in E. coli
Proteomics approach (affinity purification) for analyzing degradosome composition in E. coli
Characterizing decay in vitro, including structural studies
Defining RNA degradative activities in pathogenic bacteria (e.g., Studying tm-mediated surveillance and nonstop mRNA decay
Defining small RNA function in RNA decay
Characterizing mRNA destabilization mediated by Hfq-binding noncoding RNAs

Part II Archaea

Methods for the global analysis of mRNA stability in Archaea
In vivo and in vitro studies of degradative activities in Archaea
Structural studies of degradative activities using Archaea

Part III Eukaryotes

Dcp2/Dcp1 single-step kinetics
Dcp2/Dcp1 and DCPS activity assays
Purification and analyses of decapping activtors in Saccharomyces Reconstitution and analyses of the human Lsm complex
Analyses of deadenylation in S. cerevisiae in vitro
Deadenylation in mammalian-cell extracts
Cell-free deadenylation assays using Drosophila embryos
Cytoplasmic deadenylation assays using Xenopus oocytes
Activity and structural analyses of the reconstituted eukaryotic RNA exosome
Biochemical studies of the eukaryotic exosome
Assays of 5?-3? exonucleases in Saccharomyces cerevisiae
mRNA half-life measurements in Saccharomyces cerevisiae (all methods, including ts RNA polymerase)
Genome-wide analyses of mRNA stability in Saccharomyces cerevisiae using transcription inhibitors and microarrays (discuss different growth/stress conditions and relative half-lives)
Microscope-based cytometry to measure mRNA decay rates in large numbers of individual living Saccharomyces cerevisiae
Genome-wide analyses of mRNA stability in Trypanosomes using transcription inhibitors and microarrays (discuss different growth/stress conditions and relative half-lives)
mRNA half-life measurements in Drosophila melanogaster (all methods)
Measuring maternal transcript destabilization in Drosophila melanogaster; also mRNA destabilization during oocyte maturation and egg activation
mRNA half-life measurements in mammalian cells
Mammalian-cell mRNA decay using microarrays after block in transcription
In vivo labeling of RNA with 2,4-dithiouracil for cell-specific microarray analyses of mRNA synthesis and decay
Tracking the decay of single RNA molecules
Downregulating a decay factor and using microarrays to identify targets
Microarrays or other analyses of RNA after affinity purification or immunopurification to study RNA decay
Three-hybrid analysis to study RNA decay

Researchers in biochemistry, cell and molecular biology, genetics