k.. 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 14 3973-3977
Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome Daniel Leclerc, Pierre Melangon and Lea Brakier-Gingras* Departement de Biochimie, Universite de Montreal, Montreal H3C 3J7, Canada
Received March 11, 1991; Revised and Accepted June 19, 1991
ABSTRACT The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed,D. and Noller,H.F. (1987) Nature, 327, 389- 394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A-G and 915A-G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin. INTRODUCTION Recent work has demonstrated that specific regions of rRNA participate in the process of translation (reviewed in 1-3). Various antibiotics inhibit protein synthesis in bacteria by binding directly to rRNA (4,5, reviewed in 6). One such compound is streptomycin, an aminoglycoside drug which perturbs control of translational accuracy (7,8), by essentially abolishing the proofreading step (9-11). Streptomycin binds with high affinity to a single site on the 30S subunit of E. coli ribosome (12,13), where it primarily interacts with 16S rRNA (14,15). Cross-linking experiments have shown that streptomycin can be linked to a fragment of 16S rRNA spanning residues 892 to 917 (16), and footprinting studies indicated that bases 909, 911, 912, and predominantly bases 913 to 915, are protected by streptomycin (4). Several residues in this region are also protected
by protein S12 (see Fig. 1), which, when altered, can produce a phenotype of streptomycin resistance (21) or dependence (22).
*
To whom correspondence should be addressed
Furthermore, it has been observed that mutations at positions 912, 914 or 915 (E. coli numbering) in chloroplast 16S rRNA confer resistance to streptomycin (23,24), and mutations at position 912 have been shown to lower the response of ribosomes to streptomycin when introduced into E. coli 16S rRNA (18,19). In this study, we have introduced the nine possible single-base substitutions at positions 913 to 915 of E. coli 16S rRNA, in the plasmid pKK3535-coded rrnB operon, with the hypothesis that these substitutions would alter the response to streptomycin. Only two of these mutations were viable in E. coli cells transformed with the mutant plasmids. Ribosomes were isolated from these bacteria, as well as from bacteria transformed with a derivative of pKK3535 harbouring the 912C- U mutation (18). They were resistant to the stimulation of misreading and to the inhibition of protein synthesis induced by streptomycin, and this correlated with a reduced binding of the antibiotic. Tak-en together, these results demonstrate that the 912 to 915 region controls the binding of streptomycin to the ribosome.
MATERIALS AND METHODS Bacterial strains and plasmids Plasmid pKK3535, which contains the entire rrnB operon (25), was kindly provided by Dr Harry Noller, University of California, Santa Cruz. Plasmid pEM109, a derivative of pKK3535 with the C-U mutation at position 912 of the 16S rRNA (18), was kindly given by Dr Rolf Wagner, HeinrichHeine-Universitiit, Dusseldorf. Phagemid pDL4417, which was used for mutagenesis, is a derivative of Bluescript SK-(Stratagene) which contains the 16S rRNA gene of the E. coli rrnB operon (26). The E. coli strain K12A19, RNase I-, was the source of initiation factors and enzymes for cell-free protein synthesis assays. Strains XLI-Blue (Stratagene) and DHI (27) were used as hosts for plasmids derived from pDL4417 and from pKK3535, respectively. Strain Smr-19 is a spontaneous streptomycinresistant mutant derived from DHI, with an altered protein S12.
3974 Nucleic Acids Research, Vol. 19, No. 14 Table I. Misreading under the direction of poly(U) in the presence of streptomycin.
A
Nucleic Acids Research, Vol. 19, No. 14 3973-3977
Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome Daniel Leclerc, Pierre Melangon and Lea Brakier-Gingras* Departement de Biochimie, Universite de Montreal, Montreal H3C 3J7, Canada
Received March 11, 1991; Revised and Accepted June 19, 1991
ABSTRACT The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed,D. and Noller,H.F. (1987) Nature, 327, 389- 394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A-G and 915A-G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin. INTRODUCTION Recent work has demonstrated that specific regions of rRNA participate in the process of translation (reviewed in 1-3). Various antibiotics inhibit protein synthesis in bacteria by binding directly to rRNA (4,5, reviewed in 6). One such compound is streptomycin, an aminoglycoside drug which perturbs control of translational accuracy (7,8), by essentially abolishing the proofreading step (9-11). Streptomycin binds with high affinity to a single site on the 30S subunit of E. coli ribosome (12,13), where it primarily interacts with 16S rRNA (14,15). Cross-linking experiments have shown that streptomycin can be linked to a fragment of 16S rRNA spanning residues 892 to 917 (16), and footprinting studies indicated that bases 909, 911, 912, and predominantly bases 913 to 915, are protected by streptomycin (4). Several residues in this region are also protected
by protein S12 (see Fig. 1), which, when altered, can produce a phenotype of streptomycin resistance (21) or dependence (22).
*
To whom correspondence should be addressed
Furthermore, it has been observed that mutations at positions 912, 914 or 915 (E. coli numbering) in chloroplast 16S rRNA confer resistance to streptomycin (23,24), and mutations at position 912 have been shown to lower the response of ribosomes to streptomycin when introduced into E. coli 16S rRNA (18,19). In this study, we have introduced the nine possible single-base substitutions at positions 913 to 915 of E. coli 16S rRNA, in the plasmid pKK3535-coded rrnB operon, with the hypothesis that these substitutions would alter the response to streptomycin. Only two of these mutations were viable in E. coli cells transformed with the mutant plasmids. Ribosomes were isolated from these bacteria, as well as from bacteria transformed with a derivative of pKK3535 harbouring the 912C- U mutation (18). They were resistant to the stimulation of misreading and to the inhibition of protein synthesis induced by streptomycin, and this correlated with a reduced binding of the antibiotic. Tak-en together, these results demonstrate that the 912 to 915 region controls the binding of streptomycin to the ribosome.
MATERIALS AND METHODS Bacterial strains and plasmids Plasmid pKK3535, which contains the entire rrnB operon (25), was kindly provided by Dr Harry Noller, University of California, Santa Cruz. Plasmid pEM109, a derivative of pKK3535 with the C-U mutation at position 912 of the 16S rRNA (18), was kindly given by Dr Rolf Wagner, HeinrichHeine-Universitiit, Dusseldorf. Phagemid pDL4417, which was used for mutagenesis, is a derivative of Bluescript SK-(Stratagene) which contains the 16S rRNA gene of the E. coli rrnB operon (26). The E. coli strain K12A19, RNase I-, was the source of initiation factors and enzymes for cell-free protein synthesis assays. Strains XLI-Blue (Stratagene) and DHI (27) were used as hosts for plasmids derived from pDL4417 and from pKK3535, respectively. Strain Smr-19 is a spontaneous streptomycinresistant mutant derived from DHI, with an altered protein S12.
3974 Nucleic Acids Research, Vol. 19, No. 14 Table I. Misreading under the direction of poly(U) in the presence of streptomycin.
A
