Our goals are (1) to obtain further in vivo evidence for assembly-dependent regulation of yeast ribosomal protein synthesis, (2) to characterize biochemically the process of nuclear transport of ribosomal proteins, and (3) to characterize biochemically and functionally the assembly of yeast messenger ribonucleoprotein complexes. (1) Transcription rate, steady-state abundance, translation rate, and polyribosome distribution of mRNA for several ribosomal proteins will be measured under a condition in which the proteins can not assemble. These experiments will require cloning genes for r.p. L5 and L13, using synthetic oligonucleotides. The experiments will determine if lack of assembly causes inhibition of synthesis of these proteins and at which step inhibition occurs. (2) Nuclear localization domains of two ribosomal proteins have been identified. The effects of mutations in these regions for transport will be determined by in situ immunofluorescence, electronmicroscopic, and pulse-labeling techniques. This work will clarify the role of these domains in transport. Chemical cross-linking in conjunction with an in vitro nuclear uptake system will be used to identify nuclear proteins which mediate transport. (3) Messenger RNA-protein complexes have been formed in vitro. The role of a 5' cap and 3' polyA tail in complex formation will be determined using mRNA transcribed in vitro with or without these modifications. The identity and binding specificity of proteins in the complex will be determined by UV crosslinking and immunological analysis with antibody to a known mRNP protein. Translation in vitro will determine if the complex functions as mRNP. Assembly-dependent regulation of ribosome synthesis requires coordination between the assembly process in the nucleus and translation of ribosomal protein mRNAs in the cytoplasm. This project will provide a basis for future analysis of the connection between these events. Assembly-sensitive mechanisms appear to regulate synthesis of other housekeeping proteins, so our studies should be of general importance in determining how eukaryotic cells maintain a balanced synthesis of the individual molecules in multi-subunit structures.
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