The goal of these studies is to use the novel technique of analytical ultracentrifugation with fluorescence detection system (AU-FDS) to analyze translation complexes by detecting specific GFP- tagged proteins and mRNA. AU provides real-time analysis of the size of complexes using various wavelengths to identify biological molecules. It provides enhanced precision as to the actual size of complexes and avoids the inexactitude and the time-intensive nature inherent in secondary Western and Northern analysis. Importantly, AU-FDS allows the identification of macromolecular complexes that have not previously been visualized by standard techniques. Most critically, while other immunoprecipitation studies indicate that proteins interact, AU-FDS studies take this research to the next logical step: identifying the number, size, and composition of these protein complexes. Previously, we have used AU-FDS analysis coupled with an affinity purification of a Flag- tagged translational component (Flag-PAB1 or RPL25A-Flag) to identify from the yeast S. cerevisiae a 77S monosomal translation complex that contained mRNA, the closed-loop structural components, eIF4E, eIF4G, PAB1, and the 80S ribosome. In expansion of these studies we have used affinity purification of translation termination factor, Flag-eRF1, to identif four novel complexes, 21S, 28S, 38S, and 77S in size. The 21S/28S complexes (about 0.5 to 1.4 MDa depending on their shape) contained at least eRF1, eIF4E, eIF4G1, PAB1, HRP1, and mRNA. Many common stress granule proteins and eIF2 and eIF5 initiation factors were not present in the 21S/28S complexes. Our data suggest that the 21S/28S complexes are similar to the putative closed-loop mRNP structure. This model is consonant with the recent suggestion that HRP1 plays a role in transitioning mRNA, newly imported from the nucleus, into a translatable form. Alternatively, these complexes could be post- termination mRNP structures. In this grant we shall use the complementary techniques of AU-FDS and mass spectrometry to identify all of the components of the 21S/28S complexes. These studies will inform as to which translation initiation components, translation termination factors, and/or stress granule proteins are present in the complexes. Specific mutations in transcription, mRNA export, translation initiation, termination, and P body/stress granule formation will be used to identify at which step these Flag- eRF1 complexes are functioning. In addition, we will use a number of defined mutations in eIF4E, eIF4G, and PAB1 to investigate the structure and shape of the putative closed-loop mRNPs. Finally, the exact translational complexes bound by translational repressors, SBP1, SCD6, and NPL3, will be determined by AU-FDS using Flag-tagged versions of PAB1, eIF4E, and eRF1.
This proposal is relevant to public health by studying how protein expression is controlled. The characterization of the factors that regulate when and to what extent proteins are synthesized will illuminate the processes by which aberrant protein production leads to particular disease states.
