Mutational analysis of the C-terminal region of Saccharomyces cerevisiae ribosomal protein L25 in vitro and in vivo demonstrates the presence of two distinct functional elements.
A previous analysis of yeast ribosomal protein L25 implicated an evolutionarily conserved motif of seven amino acids near the C terminus (positions 120 to 126) in specific binding of the protein to domain III of 26 S rRNA. We analyzed the effect of various point mutations in this amino acid sequence on the capacity of the protein to interact in vitro with its binding site on the rRNA. Most of the mutations tested, including some conservative replacements, strongly reduced or abolished rRNA binding, further supporting a pivotal role for the motif in the specific interaction between L25 and 26 S rRNA. We have also determined the ability of the various mutant L25 species to complement in vivo for the absence of wild-type protein in cells that conditionally express the chromosomal L25 gene. Surprisingly, up to a fivefold reduction in the in vitro binding capacity of L25 is tolerated without affecting the ability of the mutant protein to support (virtually) wild-type rates of 60 S subunit formation and cell growth. Mutations that completely abolish recognition of 26 S rRNA, however, block the formation of 60 S particles, demonstrating that binding of L25 to this rRNA is an essential step in the assembly of the large ribosomal subunit. Using the same combination of approaches we identified an element, located between positions 133 and 139, that is indispensable for the ability of L25 to support a normal rate of 60 S subunit formation, but plays a relatively minor role in determining the rRNA-binding capacity of the protein. In particular, the presence of a hydrophobic amino acid at position 135 was found to be highly important. These results indicate that the element in question is crucial for a step in the assembly of the 60 S subunit subsequent to association of L25 with 26 S rRNA.
Department of Biochemistry and Molecular Biology, Vrije Universiteit, The Netherlands.
E A
EA
Kooi
C A
CA
Rutgers
M J
MJ
Kleijmeer
J
J
van 't Riet
J
J
Venema
H A
HA
Raué
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C031127:ribosomal protein L25