Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.
The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.
Tapio
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Department of Microbiology, Stockholm University, Sweden.
Isaksson
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LA
D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005838:Genotype; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010957:Plasmids; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism*; D000091243:Ribosomal Protein S9; D012270:Ribosomes / Q000378:metabolism; D013489:Suppression, Genetic
D003062:Codon; D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D010444:Peptide Elongation Factor Tu