Abstract
Publisher Summary Purified ribosomes are incapable of initiating the translation of messenger RNA (mRNA) unless initiation factors are added, and in the absence of these proteins, the mRNA chain does not even become attached to the ribosomes. This chapter describes the preparation of ribosomes and initiation factors for two different methods that are useful to investigate the role of initiation factors in the binding of ribosomes to mRNA. One method uses a combination of zone sedimentation and membrane filtration to detect the binding of labeled mRNA to ribosomes. While, the other is a more rapid and quantitative assay, which measures by direct electron microscopic observation, the attachment of ribosomes to DNA-bound nascent mRNA. The sedimentation of radioactive mRNA is studied on linear density gradients in which messenger RNA binds to ribosomes sediments more rapidly than free mRNA. The direct electron microscopic observation method is rapid and allows simultaneous quantitative comparison of many samples.
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