Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1.
Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.
Lapik
Yevgeniya R
YR
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607, USA
Misra
Julia M
JM
Lau
Lester F
LF
Pestov
Dimitri G
DG
D000595:Amino Acid Sequence; D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship