Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli

Abstract

Many contacts between the ribosome and its principal substrates, tRNA and mRNA, involve universally conserved rRNA nucleotides, implying their functional importance in translation. Here, we measure the in vivo translation activity conferred by substitution of each 16S rRNA base believed to contribute to the A or P site. We find that the 30S P site is generally more tolerant of mutation than the 30S A site. In the A site, A1493C or any substitution of G530 or A1492 results in complete loss of translation activity, while A1493U and A1493G decrease translation activity by >20-fold. Among the P-site nucleotides, A1339 is most critical; any mutation of A1339 confers a >18-fold decrease in translation activity. Regarding all other P-site bases, ribosomes harboring at least one substitution retain considerable activity, >10% that of control ribosomes. Moreover, several sets of multiple substitutions within the 30S P site fail to inactivate the ribosome. The robust nature of the 30S P site indicates that its interaction with the codon-anticodon helix is less stringent than that of the 30S A site. In addition, we show that G1338A suppresses phenotypes conferred by m(2)G966A and several multiple P-site substitutions, suggesting that adenine at position 1338 can stabilize tRNA interaction in the P site.

FIGURE 1.

In vivo translation activity conferred by systematic mutagenesis of 16S rRNA nucleotides contributing to the 30S A or P site. In this genetic system, translation of lacZ depends on the activity of the mutagenized ribosomes. The specific activity of β-galactosidase was quantified for each strain and normalized to the wild-type control strain, KLF10(pKF207). The 16S rRNA expressed in strain KLF10(pKF207) lacks mutations aside from the altered ASD. In each case, the data represent the mean ± standard deviation calculated from three or more independent experiments.

FIGURE 2.

Detection of mutant 16S rRNA in ribosomal fractions separated by sucrose gradient sedimentation. In each panel, primer extension products templated from wild-type and mutant 16S rRNA are indicated by arrowheads. Control reactions include those in which template was omitted (−RNA) and those in which purified 16S rRNA from wild-type E. coli strain MRE600 (wt 16S rRNA) was used as template. Ribosomal fractions (as indicated) were analyzed from strains expressing 16S rRNA containing mutation A1492U (A), A1492C (B), A1493U (C), A1493C (D), G530A (E), G530C (F), or C1192U (G).

FIGURE 3.

Effect of double, triple, and quadruple mutations of the 30S P site on translation activity. As in Figure 1, the specific activity of β-galactosidase was quantified for each strain and normalized to the wild-type control strain, KLF10(pKF207). In each case, the data represent the mean ± standard deviation calculated from three or more independent experiments.