Involvement of multiple basic amino acids in yeast ribosomal protein L1 in 5S rRNA recognition.
The role of basic amino acid residues located at the C-terminal region of the yeast ribosomal protein L1 in 5S rRNA binding was characterized in vitro and in vivo. Mutant proteins containing single or multiple amino acid substitutions were generated by site-directed mutagenesis of the L1 gene carried on a plasmid. In vitro RNP formation was examined by production of the mutant protein in the presence of the RNA molecule. The thermostability of the resultant RNP was also studied. Effects of these mutations on cell viability and ribosome assembly were characterized by transformation of a conditional null L1 yeast mutant with the mutated L1 gene expressed from the plasmid. Substitution of any one of the lysine or arginine residue did not affect significantly RNA binding in vitro or cell growth in vivo. However, several mutant proteins with substitutions of two of these basic amino acids bound RNA weakly and the RNPs were less stable. Cells expressing these mutant proteins were lethal. Theoretical structural prediction of these amino acids further provided information regarding their collective contributions to RNA recognition and to interaction between the RNP and other components of the 60S ribosomal subunit.
Yeh
L C
LC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Lee
J C
JC
D000599:Amino Acids, Diamino / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D000599:Amino Acids, Diamino; D005656:Fungal Proteins; D012341:RNA, Ribosomal, 5S; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C028546:ribosomal protein L1