Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

Abstract

Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence ⌿GG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schä fer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849 -32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and ⌿2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.Two out of the 55 UGG sequences in 23 S rRNA from Escherichia coli are universally conserved in the non-mitochondrial rRNAs of the large ribosomal subunit, UGG2251 and ⌿GG2582. The first two positions of both sequences were mutated, and the effects on ribosomal assembly and functions were analyzed (1). Mutants of the first sequence did not influence ribosomal assembly and bacterial growth but caused some defects in in vitro poly(U) translation. Those of the second sequence are characterized by a seemingly normal assembly but with severe defects in peptide synthesis (1). The ⌿GG2582 sequence is located at the "peptidyltransferase ring" (see Fig. 1). Here we analyze the peptidyltransferase (PTF) 1 activity in various systems and the translocation reaction. All mutant ribosomes were able to translocate. The helix 80 mutants show a normal puromycin reaction. In contrast, the helix 90 mutations severely interfere with the PTF activity. MATERIALS AND METHODSPlasmids and strains, the preparation of ribosomes and polysomes, and the quantification of plasmid-born 23 S rRNA have been described in the preceding paper (1).Puromycin Kinetics with AcPhe-tRNA Bound to the P-sites of Poly(U)-programmed 70 S-tRNA binding and puromycin reaction was essentially as described (2) except that the buffer conditions (20 mM Hepes-KOH (pH 7.6), 6 mM MgCl 2 , 150 mM NH 4 Cl, 4 mM ␤-mercaptoethanol, 2 mM spermidine, and 0.05 mM spermine) were kept constant in all steps (3). 70 S ribosomes were programmed with Ac[14 C]Phe-tRNA Phe (1,030 dpm/pmol, 1.5-fold excess over 70 S) and poly(U). After an incubation at 37°C for 30 min, two aliquots were filtered over nitrocellulose to determine the binding. To the remaining aliquots, puromycin was added, and the kinetics of AcPhe-puromycin formation were follow...

Mutational Analysis of Two Highly Conserved UGG Sequences of 23 S rRNA from Escherichia coli

“…It is therefore likely that the effects of the helix 90 mutations on poly(Phe) synthesis are caused by a severe distortion of the active center of the ribosomal PTF (28). The data clearly indicate that not only the single-stranded 23 S rRNA regions in the central loop of domain V are of high importance for ribosomal function but also the nucleotides at the top of helix 90.…”

“…Poly(Phe) synthesis is severely impaired. In the following paper, we were able to demonstrate that the helix 90 mutations G2581A, C2507U/G2581A, and C2507⌬/G2581A completely block the puromycin reaction, and the mutation ⌿2580C drastically impairs this reaction (28). Also C2507U reduces the AcPhe-puromycin formation by approximately 50%.…”

“…For example, the G2661C mutation of 23 S rRNA has no effect on bacterial growth until expressed in a mutant S12 background (46), but the 23 S rRNA mutation alone significantly reduces the translational efficiency (47). As shown in the following paper (28), the mutations introduced in helix 80 only slightly reduce the puromycin reaction. Therefore, none of the changed bases is essential for peptide bond formation.…”

“…Therefore, a normal tRNA binding to the A and P sites and a block in the PTF activity would be important indications for the specificity of the provoked ribosomal defect. Various functional tests were performed in this and the accompanying study (28) that require a tRNA at a specific site but differ in their ionic conditions. Therefore, 70 S ribosomes were isolated from all mutants and analyzed for binding of AcPhetRNA under the various conditions.…”

“…One of these UGG sequences is U2249 to G2251 at the helix-loop 80, and the other is ⌿2580 to G2582 at helix 90, adjacent to the PTF ring. The U (⌿) and the middle G were mutated in both sequences, and the effects on growth, expression, ribosomal assembly, and functions were studied in this and the accompanying paper (28).…”