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Methods Enzymol. 2007;429:185-201.

Molecular genetic structure-function analysis of translation initiation factor eIF5B.

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Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.


Recently, significant progress has been made in obtaining three-dimensional (3-D) structures of the factors that promote translation initiation, elongation, and termination. These structures, when interpreted in light of previous biochemical characterizations of the factors, provide significant insight into the function of the factors and the molecular mechanism of specific steps in the translation process. In addition, genetic analyses in yeast have helped elucidate the in vivo roles of the factors in various steps of the translation pathway. We have combined these two approaches and use molecular genetic studies to define the structure-function properties of translation initiation factors in the yeast Saccharomyces cerevisiae. In this chapter, we describe our multistep approach in which we first characterize a site-directed mutant of the factor of interest using in vivo and in vitro assays of protein synthesis. Next, we subject the mutant gene to random mutagenesis and screen for second-site mutations that restore the factor's function in vivo. Following biochemical and in vivo characterization of the suppressor mutant, we interpret the results in light of the 3-D structure of the factor to define the structure-function properties of the factor and to provide new molecular insights into the mechanism of translation.

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