Eukaryotic ribosome, identification

Fleischer TC, Weaver CM, McAfee KJ, Jennings JL, Link AJ (2006) Systematic identification and functional screens of imcharacter-ized proteins associated with eukaryotic ribosomal complexes. Genes Dev20( 10) 1294— 1307. doi 10.1101/gad.1422006... 

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (elF2 GTP Met-tRNAjMet) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target. 

The initiation process differs for prokaryotes and eukaryotes (Table 15.3). In bacteria, the initiating methionyl-tRNA is formylated, producing a formyl-methionyl-tRNAf that participates in formation of the initiation complex (Fig. 15.9). Only three initiation factors (IFs) are required to generate this complex in prokaryotes, compared with the dozen or more required by eukaryotes. The ribosomes also differ in size. Prokaryotes have 70S ribosomes, composed of 308 and 50S subunits, and eukaryotes have SOS ribosomes, composed of 40S and 60S subunits. Unlike eukaryotic mRNA, bacterial mRNA is not capped. Identification of the initiating AUG triplet in prokaryotes occurs when a sequence in the mRNA (known as the Shine-Dalgamo sequence) binds to a complementary sequence near the 3 -end of the 16S rRNA of the small ribosomal subunit. 

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