Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8. Generation of a mutant lacking ribosomal protein S17.
S17 is a primary rRNA-binding protein which has been implicated in ribosome assembly and translational fidelity. We describe the generation and biochemical characterization of an S17 minus ribosomal mutant, a ribosomal protein-lacking mutant obtained in Thermus thermophilus HB8. The S17 mutant was obtained by insertional inactivation of the target gene with the kanamycin adenyl transferase (kat) gene, making use of a Thermus-Escherichia shuttle vector and the natural ability of Thermus to transform. In the final construct used to transform Thermus cells, the S17 coding region was replaced with the kat gene cloned in-frame with the first three amino acids of S17. Hence, in vivo transcription of the kat gene was under the control of the ribosomal operon promoter. As in Escherichia coli, the Thermus S17 mutant exhibited a temperature-sensitive phenotype. Two-dimensional PAGE, Western blot, and ELISA confirmed the absence of S17 from the mutant ribosomes. Sucrose-gradient profiles of mutant cells showed a clear separation and normal proportions of 50S and 30S subunits and a normal ratio between them. In addition, the S17 mutant showed the presence of a 20S peak representing assembly-defective particles. The successful re-incorporation of protein S17 into the mutant ribosomes was demonstrated when reconstitution with isolated S17 was performed at 60 degrees C.
Simitsopoulou
M
M
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Berlin, Germany.
Avila
H
H
Franceschi
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F
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D007612:Kanamycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012269:Ribosomal Proteins / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D016963:Thermus thermophilus / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
D000900:Anti-Bacterial Agents; D009841:Oligonucleotides; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D013395:Sucrose; D007612:Kanamycin; D004247:DNA