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Abstract
This chapter describes the implications of ribosome loading onto messenger Ribonucleic acid (mRNA) species on translational control. Analysis of the association of an mRNA species with ribosomes can give great insight into: (1) occurrence of translational control of expression of the mRNA and, (2) possible mechanisms of regulation. Regulation of expression of the protein product of a gene can occur at several levels. Translation of an mRNA molecule occurs in a multimeric structure, the polysome that consists of multiple ribosomes arrayed along the length of the mRNA. Polysomes are readily separated according to the number of associated ribosomes by velocity sedimentation through a gradient of sucrose, which serves to stabilize the boundaries of the sedimenting zone. This chapter describes methods for the preparation of cellular extracts, the display of ribonucleoprotein particles, and polysomes by sucrose gradient centrifugation, and the detection of mRNA species of interest. The chapter discusses in detail about the polysome profiles and translational control. The chapter also explains concepts related to the extraction and display of polysomes RNA analysis by northern blots, elaborating upon the analysis of Ribonuclease protection, assay, and hybridization.
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