A single base change at 726 in 16S rRNA radically alters the pattern of proteins synthesized in vivo.
A single base change in 16S rRNA (C-726 to G) was constructed by site-directed mutagenesis and cloned into the multicopy plasmid pKK3535 (generating pKK726G) which contains the complete rrnB operon from Escherichia coli. The mutant 16S rRNA was found predominantly in the 30S subunit fraction but was present in the 70S ribosomes. Protein analyses of the free 30S subunits revealed a decrease in the levels of ribosomal proteins S2 and S21 while the composition of the 70S ribosomes was as the wild-type. Transformants of pKK726G were temperature sensitive for growth, although the mutant ribosomes themselves were translationally active in vivo at 37 and 42 degrees C. Two-dimensional gel electrophoresis of the proteins translated in vivo revealed an altered protein profile which included novel proteins, changes in the levels of normal proteins, and the presence of heat shock proteins (HSPs) at 30 degrees C. Inactivation of the host encoded wild-type ribosomes coincided with a significant decrease in the synthesis of the HSPs. We therefore believe the induction of the HSPs to be a secondary response by the cells to the presence of the abnormal proteins.
Prescott
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Section of Biochemistry, Brown University, Providence, RI 02912.
Dahlberg
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D001483:Base Sequence; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature; D014169:Transformation, Bacterial
D006360:Heat-Shock Proteins; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S