The central proposal of this project is that the aliphatic polyamines, putrescine, spermidine, and spermine, may exert some regulatory role in eukaryotic gene expression in the nucleolus of eukaryotic organisms. The specific objectives are: (1) to investigate the molecular mechanism of these compounds in mediating the phosphorylation of a specific nucleolar, nonhistone chromosomal (NHC) protein; (2) to characterize the interactions of a specific 70,000-Mr NHC protein, whose phosphorylation is polyamine-dependent, with the structure and in vitro transcription of ribosomal RNA (rRNA) genes; and (3) to determine whether polyamine control of rRNA gene expression can be demonstrated in nucleoli of the slime molds, Physarum polycephalum, Dictyostelium discoideum, and in nuclei of rat liver cells. In earlier projects we demonstrated a polyamine-dependent NHC protein kinase in nucleoli of P. polycephalum. This kinase phosphorylated a unique NHC protein of 70,000 Mr which stimulated specific rRNA gene transcription in an isolable minichromosome derived from nucleoli. This 100S minichromosome contained RNA polymerase I, palindromic rDNA, the 70,000 Mr NHC phosphoprotein and a complement of other uncharacterized proteins. The 70,000-Mr NHC phosphoprotein bound with high specificity to rDNA and to restriction endonuclease fragments of rDNA containing the central quarter of the rDNA palindrome. The phosphorylated state of the phosphoprotein determined its capacity to stimulate rRNA gene transcription and to bind to rDNA. This project proposes to purify the polyamine-dependent NHC protein kinase. The nature of the polyamine-dependency will be investigated with arylazido-polyamine analogs, a new class of photoaffinity-labeling reagents. The mechanism of stimulation of transcription by the 70,000-Mr NHC phosphoprotein will be studied with ATP gamma S to probe for its role in new rRNA chain initiation. The precise location of phosphoprotein-binding to rDNA and to the minichromsome during stimulated transcription will be determined with heterobifunctional, photoaffinity crosslinking agents and restriction endonuclease mapping of rDNA. The possible identity of the 70,000 Mr NHC protein with ornithine decarboxylase, as suggested by preliminary work, will be probed by immunological methods and by characterization of both proteins.
NIH ExPorter
NIH ExPorter