A long-standing question in biology concerns the in vivo assembly of ribosomes, which are the biological machines that translate the genetic code into proteins. In this project, early steps in biogenesis of the small ribosomal subunit will be studied by electron microscopic visualization of nascent ribosomal RNA transcripts in the yeast, Saccharomyces cerevisiae. Five reproducible assembly intermediates for the small subunit are discernible on nascent transcripts, beginning with formation of a small 5' particle and culminating in compaction of the entire 18S rRNA sequence into a large terminal knob (the pre-small subunit), which is released co-transcriptionally by RNA cleavage. Remarkably, an intermediate assembly step involves formation of three kinked nodes of RNP formation in the 18S RNA, which map approximately to the three known domains of 18S rRNA and which collapse onto each other during a process that dramatically compacts the length of the RNA into the large terminal knob. These events are reminiscent of findings from bacterial ribosome reconstitution studies, in which it was shown that the three subdomains of the small subunit (body, platform, head) are autonomous assembly domains. Thus, the hypothesis that will be tested is that the three nodes of RNP formation represent assembly of ribosomal proteins onto the three structural domains of 18S RNA, which will be the first test of this assembly pathway in vivo. A combination of yeast genetics and electron microscopy will be used to test the hypothesis by depletion of early-assembling ribosomal proteins and by insertion of rRNA expansion sequences to test for predicted effects on the size or spacing of nodes. Specific proteins will be localized in the structures using antibody or cytochemical labeling for EM. This project will help elucidate the process of eukaryotic ribosome biogenesis in living cells, and represents a new experimental handle on this extremely important and complex biological process. It will also present training opportunities for students, including an annual summer research internship for an undergraduate from an underrepresented minority group.
