We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycindependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.Keywords: ribosomal protein S12; streptomycin; point mutation; cell-free protein synthesis.The antibiotic streptomycin inhibits protein synthesis and causes misreading during translation. Ribosomal protein mutations in Escherichia coli have been found to confer resistance to streptomycin [1,2]. These mutations frequently exist in the ribosomal protein S12, encoded by rpsL, and result in streptomycin resistance [3] or streptomycin dependence [4]. The phenotypes were attributed to the mutations in the S12 protein by Funatsu et al. [5,6]. The streptomycin-resistance mutations in the ribosomal proteins S4 and S5 confer ribosomal ambiguity (ram) phenotypes, and cause a decrease in the translational accuracy [7,8]. In the 1980s, mutations conferring streptomycin resistance were found in the 16S rRNA of bacteria and chloroplasts (rRNA Mutation Database, located at http://www_fandm. edu). Many of them were near the 530-loop, which has been proposed to form a pseudoknot structure [9], and were stabilized by the S12 protein, as shown in a footprinting study of the 30S ribosomal subunit [10]. A genetic analysis of the 16S rRNA mutations and chemical probing for each 16S rRNA mutation in the S12 mutant strains demonstrated that the streptomycin resistance was achieved by a lower affinity for streptomycin, and all of the mutations gave rise to conformational changes in the rRNA [11,12]. Studies of streptomycin resistance and dependence in 23S rRNA mutations have shed light on the relationship between accurate decoding and GTP hydrolysis by EF-Tu [13][14][15]. Although the pseudoknot structure and the S12 ribosomal protein are clearly responsible for translational accuracy, the streptomycin did not bind to th...
Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.
We analyzed the effect of nine 'rare' codons (AGA, AGG, AUA, CCC, CGA, CGG, CUA, GGA, and UUA) on gene expression in an Escherichia coli coupled transcription/translation cell-free system, in comparison with a cell-based expression system. Each reporter gene contained five consecutive repeats of a rare codon, or in some experiments, three consecutive repeats. The cell-free expression of the genes bearing the codons CGA, CUA, GGA, and UUA was not affected, although these codons, except for GGA, were inefficiently translated in E. coli cells. Translation of the remaining five codons (AGA, AGG, AUA, CCC, and CGG) was severely reduced in both systems, and was remarkably facilitated in the cell-free system based on an S30 extract from the E. coli cells overproducing 'minor' tRNAs for these codons.
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