In vivo and in vitro analysis of structure-function relationships in ribosomal protein L25 from Saccharomyces cerevisiae.
We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.
Rutgers
C A
CA
Biochemisch Labatorium, Vrije Universiteit, Amsterdam, The Netherlands.
Schaap
P J
PJ
van 't Riet
J
J
Woldringh
C L
CL
Raué
H A
HA
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D015825:Chromosomes, Fungal; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D016297:Mutagenesis, Site-Directed; D012335:RNA, Ribosomal / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013329:Structure-Activity Relationship
D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C031127:ribosomal protein L25