title: Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells.
abstract: Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation.
authors.affiliation: Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shigeno
authors.affiliation: Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Uchiumi
authors.affiliation: Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan. Electronic address: nomurat@shinshu-u.ac.jp.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Nomura
mesh_terms: D001089:Arabinose / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D054657:Ribosome Subunits / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C031249:ribosomal protein L6; D001089:Arabinose; D020558:GTP Phosphohydrolases
keywords: 45S particle; Biphasic growth; Immature 50S subunit; L6; Ribosome assembly