23S rRNA positions essential for tRNA binding in ribosomal functional sites

Abstract

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.

Figure 1

(A) The chemical structure of the Q-nucleoside present in the anticodon loop of tRNA^Tyr. (B) An outline of an experimental scheme leading to formation of a complex between N-acetyl-aminoacyl-tRNA and the large ribosomal subunit and capturing the complex on the resin. N-Acetyl-tyrosyl residue is shown as a solid circle, biotin as a triangle, the large ribosomal subunit as a gray oval, and the avidin resin as a hatched semicircle.

Figure 2

(A) Binding of N-Ac-Tyr-tRNA_Biot^Tyr to the large ribosomal subunit in the fragment reaction conditions. The amount of bound tRNA was estimated by nitrocellulose filter binding. Open circles represent binding in the absence of sparsomycin; solid circles show binding in the presence of 0.1 mM sparsomycin. (B) Trapping of the complex of large ribosomal subunits with N-acetyl-aminoacyl-tRNA on the avidin resin. ³²P-labeled 50S subunits were incubated with nonbiotinylated N-Ac-Tyr-tRNA, N-Ac-Tyr-tRNA_Biot, or N-Ac-Tyr-tRNA_Biot in the presence of sparsomycin and passed through the avidin resin, and, after washing, the captured material was eluted and counted.

Figure 3

Analysis of base modifications of 23S rRNA in randomly modified 50S ribosomal subunits before and after selection on the avidin resin. (A) Modification with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide. (B) Modification with dimethyl sulfate. (C) Modification with kethoxal. rRNA was extracted from the 50S subunits, and modifications were localized by primer extension. Lanes: 1, 23S rRNA from the unmodified 50S subunits; 2, modified, unselected 50S subunits; 3, modified subunits complexed in the presence of sparsomycin with N-Ac-Tyr-tRNA and captured on the avidin resin. Dideoxy sequencing lanes corresponding to RNA bases U, A, C, and G are shown. Dots show the reverse transcriptase “strong stop” bands whose intensity is decreased in the selected samples.

Figure 4

(A) Puromycin-dependent release of radioactivity from the N-Ac-[³H]Tyr-tRNA_Biot^Tyr complexed with the large ribosomal subunit and captured on the avidin resin. Biotinylated N-Ac-[³H]Tyr-tRNA was incubated with 50S subunits and captured on the resin in the absence of sparsomycin (see Materials and Methods). The resin was saturated at 0°C with the FR/M buffer containing either no puromycin, 0.2 mM puromycin, or 0.2 mM puromycin and 8 mM chloramphenicol. After 10 min of incubation, eluted radioactivity was counted and plotted as percentage of the total radioactive tRNA captured on the column. (B) Puromycin-dependent release of large subunits from the complexes with tRNA captured on the avidin resin. ³²P-labeled 50S subunits were complexed with N-Ac-Tyr-tRNA_Biot^Tyr, captured on the avidin resin, and incubated 10 min at 0°C in the FR/M buffer containing either no puromycin, 0.2 mM puromycin, or 0.2 mM puromycin and 8 mM chloramphenicol. The column was washed with the FR/M buffer, and 50S subunits remaining on the column after this treatment were eluted and counted. (C) Analysis of base modifications of 23S rRNA in randomly modified 50S ribosomal subunits eluted by puromycin from the complex with N-Ac-Tyr-tRNA_Biot^Tyr captured on avidin resin. Lanes: 1, rRNA from the unmodified 50S subunits; 2, unselected 50S subunits modified with cyclohexyl-3-(2-morpholinoethyl)carbodiimide; 3, rRNA from modified subunits complexed with N-Ac-Tyr-tRNA_Biot^Tyr, captured on the avidin resin, and eluted by puromycin-containing buffer. U-specific dideoxy sequencing lane is shown. Dots indicate the reverse transcriptase “strong stop” bands whose intensity is decreased in the selected samples.

Figure 5

Summary of the positions in domain V of 23S rRNA whose modification interfere with binding of N-acetyl-aminoacyl-tRNA in the P site (•) or with puromycin reaction (▴). Possible interactions of individual P site residues with the CCA-end and aminoacyl moiety of N-acetyl-aminoacyl-tRNA are shown by dotted lines.