The synthesis of proteins from 20 or so constituent amino acids according to a strictly defined code with an accuracy of better than 1 in 10,000 at most loca tions is arguably the most complex task performed by cells. Protein Synthesis collects together methods and protocols covering a range of different approaches towards understanding how the cellular machinery accomplishes this task and how these ftinctions might be harnessed by the biotechnology industry to generate novel and useful proteins. The era in which the components of the translational machinery were being catalogued is over. This volume gathers together protocols that focus on preserving and describing the dynamic function as closely as possible. The need to understand exactly how ribosomes are positioned on messages or where tRNA molecules, translation factors, or control proteins are bound, has been appreciated by many of the authors. Several chapters that explore the fidelity and processivity of translation reflect this belief. Moreover, the fundamental importance of rRNA at the heart of the ribosome is a strong theme in a number of the protocols. These articles include in vitro and in vivo systems from bacterial, fungal, plant, and animal systems. Overall, Protein Synthesis might be characterized by the novelty of the approaches employed to illuminate the inner workings of the protein synthetic machinery as well as by the inventiveness of the attempts to harness these reactions for biotechnological applications.

Klappentext

This book presents a collection of molecular biological methods specific to protein synthesis. Chapters open with a discussion of basic background information and strategy which is then complemented by comprehensive methodological details. The book is divided into seven significant areas that cover all of the research techniques required by both experienced researchers and newcomers to the field of protein synthesis, and will prove to be an invaluable reference source on the benchtop of many protein laboratories.

Inhalt
Analyses of Ribosome Distribution During In Vitro Translation.- Allele-Specific Priming in the Mapping of rRNA.- Permeabilized Mammalian Cells as a System for Protein Synthesis.- The Measurement of Processivity Errors in Protein Synthesis.- A Biosynthetic Approach for the Incorporation of Unnatural Amino Acids into Proteins.- The Analysis of Translational Activity Using a Reporter Gene Constructed from Repeats of an Antibody-Binding Domain from Protein A.- In Vitro Engineering Using Acyl-Derivatized tRNAs.- In Vitro Engineering Using Synthetic tRNAs with Altered Anticodons Including Four-Nucleotide Anticodons.- Analysis of rRNA Function Using Specialized Ribosomes.- Determination of the Peptide Elongation Rate In Vivo.- The Measurement of Missense Errors During In Vivo Protein Synthesis.- Heelprinting Analysis of In Vivo Ribosome Pause Sites.- Analysis of elF-2? Kinases in Yeast.- Continuous-Flow Cell-Free Translation, Transcription-Translation, and Replication-Translation Systems.- Analysis of Translational Activity of Extracts Derived from Oocytes and Eggs of Xenopus laevis.- A Fractionated Reticulocyte Lysate System for Studies on Protein Synthesis Initiation Factors.- Measurement of Ribosomal Accuracy and Proofreading in E. coli Burst Systems.- Measurement of Rate of Protein Synthesis In Vitro.- In Vivo Mutational Analysis of Ribosomal RNA in Saccharomyces cerevisiae.- Genetic Selection of rRNA Mutations.- Toeprinting Assays.- Mapping of Pseudouridine Residues in RNA to Nucleotide Resolution.- Chemical and Enzymatic Probing of Antibiotic-Ribosome Complexes.- Protein Engineering with Nonstandard Amino Acids.- Internal Ribosome Entry Sites Tests with Circular mRNAs.- Gel Retardation and UV-Crosslinking Assays to Detect Specific RNA-Protein Interactions in the 5? or3? UTRs of Translationally Regulated mRNAs.- Translational Control by Repressor Proteins Binding to the 5?UTR of mRNAs.- Identification and Analysis of Frameshift Sites.- Synthesis and Site-Specific Binding of Thioated tRNAs to Probe Ribosome-tRNA Interactions.