The accessibility of yeast ribosomal protein L1 as probed by proteolysis and site-directed mutagenesis is different in intact 60 and 80 S ribosome.
Accessible regions of protein L1 in intact 60 and 80 S ribosomes from Saccharomyces cerevisiae were first detected by controlled proteolysis. The N-terminal region of L1 in either 60 S or 80 S particles, was inaccessible to proteases, but the central and C-terminal regions were accessible. The accessibility of the central region differed depending on the ribosome state. These regions were further examined by determination of the chemical reactivity of specific cysteine residues introduced into these regions by site-directed mutagenesis. All cysteine mutant proteins were capable of binding yeast 5 S rRNA in vitro and the ribosomes containing the mutant proteins were functional in vivo. Residues Cys-257 and Cys-275 were modified in both the 60 and 80 S ribosomes but the modification rates were different in the two ribosome states. Both residues Cys-62 and Cys-286 were inaccessible in 80 S or 60 S ribosomes. Taken together, the present study identified several accessible regions of L1 in intact ribosomes and further showed that the accessibility of some of the regions was altered upon ribosomal subunit association. The most likely interpretation of these results is that the conformation of the ribosomal protein L1 was altered upon ribosomal subunit association.
Lee
J C
JC
Department of Biochemistry, University of Texas Health Science Center, San Antonio, 78284, USA.
Turgeon
C L
CL
Yeh
L C
LC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003488:Cyanogen Bromide; D003545:Cysteine; D017931:DNA Primers; D010450:Endopeptidases; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; D003545:Cysteine; D003488:Cyanogen Bromide