Reconstitution of Functional 50S Ribosomes from in Vitro Transcripts of Bacillus stearothermophilus 23S rRNA

Abstract

In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytically active 50S ribosomal subunits with an efficiency only 3-4-fold lower than that of natural 23S rRNA. Thus, post-transcriptional modifications in 23S rRNA are not essential for the assembly or function of the 50S subunit of the ribosome. This reconstitution sytem has been used to characterize the peptidyl transferase activity of site-directed mutations in 23S rRNA at positions G2252, U2506, U2584, and A2602 (Escherichia coli numbering), demonstrating its potential for the analysis of the role played by 23S rRNA in the function of the 50S subunit of the ribosome.

Peptidyl transferase antibiotics perturb the relative positioning of the 3′-terminal adenosine of P/P′-site-bound tRNA and 23S rRNA in the ribosome

“…Three-dimensional histograms showing the relative yields of rRNA cross-links obtained in the presence and absence of antibiotics for deacylated-tRNA-ribosome complexes (A) and N-Ac-PhetRNA-ribosome complexes (B)+ The results are summarized for a sample without antibiotics (Control) and for samples with the antibiotics sparsomycin (Spar, 0+02 mM), pristinamycin IA (PIA, 0+1 mM), chloramphenicol (Cam, 1 mM), pristinamycin IIA (PIIA, 0+1 mM), pristinamycin IA ϩ IIA, gougerotin (Goug), lincomycin (Linc), and spiramycin (Spira) (all at 0+1 mM)+ In the color coding, blue indicates a significant decrease in cross-linking yield relative to the control samples, whereas red denotes a significant increase+ Error limits are listed in Table 3+ linking to the RNA backbone is less likely to cause primer extension stops+ Although we cannot exclude the possibility that some of the cross-linked sites lie in intermediate states occupied during entering or leaving of the P9 site, we conclude, nevertheless, that all these nucleotides play an important role in the peptidyl transferase reaction+ Each of the identified cross-linked nucleotides except C2601/A2602 falls within the earlier designated fragments F1, F2, and F4 (Wower et al+, 1995)+ On the basis of the primer extension method, we cannot exclude the possibility that additional cross-links remain undetected which produce bands that comigrate with control bands arising, for example, from posttranscriptionally modified nucleotides (Fig+ 6)+ Nevertheless, the results correlate with an earlier finding that removal of the 39-terminal adenosine from P-site-bound deacylated tRNA leads to enhanced chemical reactivities of U2506 and U2584/2585 (Moazed & Noller, 1989)+ It is also consistent with the observation that the chemical reactivity of A2602 is enhanced when an N-blocked aminoacyl group is coupled to the P-site-bound tRNA (Moazed & Noller, 1989)+ Damage-selection experiments with the 39-terminal fragment of N-Ac-Tyr-tRNA provide additional support for the involvement of U2506 and U2585 in P9-substrate binding (Bocchetta et al+, 1998)+ Furthermore, the functional importance of U2506 and U2584/U2585 is strongly underlined by the demonstration that all possible mutations at these nucleotides except U2584C produce lethal growth phenotypes in E. coli, and generally result in reduced levels of peptidyl transferase activity in isolated ribosomes (Porse et al+, 1996;Green et al+, 1997;Green & Noller, 1999)+ All of the antibiotics tested except sparsomycin have been footprinted on the 23S rRNA of free ribosomes, where they induce multiple changes in the chemical reactivities of nucleotides within the peptidyl transferase loop region that includes the cross-linked positions A2062, U2506, and U2585 (Moazed & Noller, 1987;Douthwaite, 1992;Rodriguez-Fonseca et al+, 1995;Porse & Garrett, 1999)+ Moreover, for some of the drugs, FIGURE 6. Primer extension analyses on 23S rRNA showing the effects of selected antibiotics on the cross-linking sites of N-Ac-Phe-[ 32 P](2N 3 A76)tRNA at U2506, U2584/U2585, and C2601/A2602 (within the fragments F19 and F29) using primer EC2621 (A) and at A2062/C2063 (within F...…”

Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance

“…Importantly, although the collective importance of rRNA modifications for protein synthesis has been shown (11), rsmG mutants developed even in the genetic background of ksgA when selected for low-level streptomycin resistance (e.g., KO-924 to KO-926) ( Table 1), suggesting that ksgA and rsmG mutations can coexist without a loss of cell viability. Allowance of coexistence was confirmed by transformation (using KO-847 as the recipient and KO-750 as the donor DNA), in which ksgA rsmG transformants readily developed (e.g., KO-927) ( Table 1).…”

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

“…Nucleotide substitutions at A2451 were incorporated into the plasmid pBST7-23S by using the QuikChange protocol (Stratagene) and confirmed by sequencing. B. stearothermophilus 50S subunits were reconstituted from in vitro transcripts of 23S rRNA as described (27).…”

“…To determine the effects of altering A2451 or G2447 on catalysis more directly, we measured the activity of mutant ribosomes in assays that measure single-turnover peptidyl transfer events. Pure populations of 2451 mutant 50S ribosomal subunits were reconstituted from in vitro transcribed B. stearothermophilus 23S rRNA (27), and their peptidyltransferase activity was measured in two different single-turnover assays.…”