Ribosome biogenesis in eukaryotes is a complex process that involves more than 170 trans-acting proteins. Many of these trans-acting factors are predicted to be enzymes that are important for assembly. One of these proteins, Saccharomyces cerevisiae Nog1p, is a nucleolar member of the Obg/CgtA family of monomeric GTPases. Depletion of Nog1p leads to defects in 60S ribosomal subunit assembly, rRNA processing, and 60S ribosomal subunit nuclear export, indicating that Nog1p is critical for the biogenesis of the 60S ribosomal subunit. This dissertation focuses on the characterization of mutants and truncated variants of Nog1p. Several mutations in conserved residues of the GTP-binding domain of Nog1p affect cell growth and 60S assembly, but do not affect the association of Nog1p with the pre-60S particle. A mutation in the GTP-binding domain, nog1P176V, was found to modestly affect the steady-state levels of rRNA precursors and the protein levels in pre-60S complexes. Studies utilizing truncated variants of Nog1p revealed that, of the 647 amino acids that comprise Nog1p, both residues 1-126 and the highly acidic region between 456 and 479 are necessary for function, but not for ribosome association. In contrast, however, amino acids 347-456 are critical for 60S ribosome association. Additionally, the last 168 amino acids of Nog1p (479-647) contribute to, but are not critical for Nog1p function. Clustered charge-to-alanine mutagenesis studies revealed several amino acids in both the N- and C-termini that are also important, but not critical for Nog1p function. Together these data suggest that Nog1p makes multiple contacts with interacting partners in the cell, and that these interactions are important for optimal Nog1p function.Nissan TA, Bassler J, Petfalski E, Tollervey D, Hurt E. 2002. 60S pre-ribosome formation ... Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: ... 782: 2771-2777 a#39;. Seeburg PH, Colby WW, Capon DJ, Goeddel DV, Levinson AD. 137 .
|Title||:||Characterization of Saccharomyces cerevisiae Nog1p: A nucleolar putative GTPase critical for 60S ribosomal subunit biogenesis|
|Author||:||Jennifer Lynn Fuentes, University of Michigan|