Effects Of Tra1 Gene Mutations On Cell Growth And Gene Expression

Growth of eukaryotic cells is a highly complicated issue and is closely related to changes of nutrient composition and to the environmental variations. Modulation of cell growth is underlied by complex but coordinated gene expression and regulation. Ribosomes are the cellular factories responsible for protein synthesis in supporting cell growth. To emphasize the significance of driving most of the cellular energy on the synthesis of ribosome, ribosomal biogenesis invokes utilization of three eukaryotic RNA polymerases, RNA polâ… , polâ…¡, and polâ…¢. Although study on the regulation of ribosomal RNA genes is one of the intensively focused topics, it will not be possible to fully elucidate the mechanism if the investigation is out of the context of the expression and regulation of other ribosomal components and the control of cell growth. Tral protein is a common subunit of SAGA and NuA4 complexes, two well-studied transcriptional coactivators in yeast with its homologues also reported in human. Biochemical and genetic experiments suggest that Tral may be involved in the transcriptional control of ribosomal protein genes and ribosomal RNA genes through NuA4 complex as well as expression of stress-induced genes through SAGA complex. In this study, we used Tral 3355 mutation, Tral 3285+3375 double mutation yeast strains to measure growth curves in different growth conditions; extracted total RNA and examined the expression of different target genes (His3,His4,RPS7A,Actl,Scrl) by Northern analysis. We found that Tral 3355 mutation and Tral 3285+3375 double mutation strains have distinct effects on the expression of ribosomal RNA,His3,His4 and Actl genes. Unexpectedly, we also found significant accumulation of small RNAs with sizes similar to tRNAs in Yral 3355 mutation and Tral 3285+3375 double mutation strains. These results lay a foundation to comprehensively analyze the effects of Tral mutation on genome-wide gene expression by microarray. To determine the tegrity of SAGA and NuA4 complexes in the mutant backgrounds, we also tagged some subunits of SAGA and NuA4 complexes for the subsequent experiments by co-immunoprecipitation and western analyses. With these materials in hands, it is possible for us to deeply investigate the roles of Tral protein on ribosome biogenesis and on the coordinated regulation of cell growth in eukaryotes.