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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.
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1 Analyses of Ribosome Translation
Distribution
During In Vitro
Sandra L. Wolin 1. Introduction For studies of translational regulation, it is often important to determine the distribution of ribosomes along a particular mRNA species. Knowledge of the exact positions of stalled ribosomes may aid in the identification of features of mRNA sequence and structure that result m ribosome stalling. We therefore devised an assay to allow the determination of ribosome distribution on an mRNA with single nucleotide precision (1). This assay can be used to map the positions of ribosomes during in vitro translation of any mRNA for which a full-length cDNA clone is available Since development of this assay, it has been used to show that ribosomes frequently pause at initiation and termination codons (1,2), to examine the arrest of translation by signal recognition particle (1,3,4) and to demonstrate that ribosomes pause at the site of a pseudoknot required for frameshifting (5). The assay has also been adapted to monitor the positions of paused ribosomes on abundant mRNAs m vivo (6,7). The assay is based on the fact that ribosomes protect 30-35 nucleotrdes of mRNA from ribonuclease digestion (reviewed in ref. 8). Extracts undergoing translation are treated with nuclease to digest mRNA fragments that are not protected by ribosomes. If ri