Research in this laboratory is directed at understanding the cellular mechanisms responsible for induced differentiation of Friend murine erythroleukemia (MEL) cells. Recent results demonstrate that: 1) transformation of these erythroblasts directly affects translation initiation; and 2) exposure to inducers of differentiation rapidly decreases translation rate and this decrease precedes differentiation. This and other abundant evidence indicate that translation is a significant regulator of gene expression in transformed cells. The experiments outlined in this proposal will determine the role of translational regulation of gene expression in transformation and differentiation of MEL cells. Inducers are shown to affect a previously undescribed site for translational regulation where MRNA accumulates as monosomes and only slowly progresses to translating polysomes. The accumulation of monosomes appears to be due to """"""""stalling"""""""" at the initiating AUG codon of MRNA. The experiments outlined in this proposal will determine the precise step in translation affected by inducers by """"""""footprinting"""""""" the interaction of ribosomes and MRNA and analyzing the tRNAs and peptides present in the stalled monosome. Factors which regulate stalling will be analyzed by developing an in vitro assay for this event and by purification and further characterization of two ribosomal proteins which are specifically absent from these stalled monosomes. Translation initiation rate will be selectively increased or decreased in MEL cells utilizing a recently described genetic approach. This will determine the role of translation initiation in transforming these cells and in their induced differentiation. The results of these experiments will provide insight into eukaryotic growth regulation and translations) regulatory mechanisms.
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