The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center

Abstract

Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.

Figure 1.

Structural organization of the inner core PTC nucleotides. (A) Secondary structure of the central loop of domain V of T. aquaticus 23S rRNA (52). The five inner core residues (A2602, U2585, U2506, A2451 and C2063) as well as the ‘second layer’ residue A2450 are highlighted in bold and are colored. (B) The 3D architecture of the PTC structure using the same color code as in (A). The hydrogen bonding interactions of the non-Watson-Crick base pair A2450–C2063 are shown. The position of the nitrogen atom of the attacking α-amino group of the Phe-tRNA in the A-site is depicted as green sphere. The figure was generated from to the T. thermophilus 70S atomic coordinates (pdb files 2WDK and 2WDL) (32).

Figure 2.

Effects of A2450–C2063 base pair modification on in vitro translation, peptide bond formation and pept-tRNA drop-off. (A) Schematic illustration of the A–C wobble base pair (middle) and the introduced nucleotide modifications at positions A2450 (left) and C2063 (right). (B) In vitro translation activities of ribosomes with A2450 or (C) with C2063 modifications. The amount of polyphenylalanine produced by reconstituted ribosomes (in cpm or in phenylalanine per 70S ribosome) is plotted as a function of time. (D) The amount of extracted dipeptidyl product (N-acetyl-Phe-[³H]Phe) utilizing N-acetyl-[³H]Phe-tRNA^Phe and [³H]Phe-tRNA^Phe as reaction substrates formed on reconstituted ribosomes carrying the wt oligo was compared to the product obtained with ribosomes carrying an abasic site at position 2450 (aba). Product formation of wt ribosomes (∼0.1 pmol of N-acetyl-Phe-[³H]Phe per 1 pmol of 70S) was taken as 1.0 (E) The same ribosomal complexes as in (D) were used to quantify the fraction of N-acetyl-Phe-[³H]Phe-tRNA^Phe retained on the ribosome after peptide bond formation via filtration through nitrocellulose membranes. The amount of ribosome-bound dipeptidyl-tRNA on reconstituted wt ribosomes was taken as 1.00. Background values of retained [³H]Phe-tRNA^Phe on the nitrocellulose membrane in the presence of native 30S subunits alone were subtracted (∼0.025 pmol per 1 pmol of 30S subunits). In (A–E) the values represent mean and standard errors of at least three independent experiments.

Figure 3.

EF-G GTPase activities triggered by ribosomes containing modifications at A2450 or C2063. (A) EF-G catalyzed GTP hydrolysis induced by reconstituted ribosomes with disrupted (abasic 2450 or 2063, aba) or an intact A2450–C2063 base pair (wt). The amount of GTP hydrolyzed over time is plotted. The GTP input in a single experimental time point was 625 pmol. (B) Products of the ribosome-dependent uncoupled EF-G GTPase reactions were separated via thin-layer chromatography and visualized by phosphor imaging. Values were obtained by quantification of the released inorganic phosphate (Pi) and represent the mean and standard errors of at least three independent time course experiments. Background values (amount of product in the presence of 30S subunits alone) were subtracted from every data point. In the absence of 30S particles, reconstituted 50S subunits do not trigger detectable GTP hydrolysis on EF-G (9).

Figure 4.

Effects of A2450–C2063 base pair disruption on the length of the produced poly(Phe) peptides. (A) A representative TLC plate with poly([¹⁴C]Phe) peptides synthesized in poly(U)-dependent translation reactions is shown. Lanes show synthesized poly([¹⁴C]Phe) peptides and unincorporated [¹⁴C]Phe after translation using ribosomes reconstituted with the wt oligo (wt), ribosomes reconstituted with the oligo carrying an abasic site at 2450 (aba2450), native E. coli 70S (70S) as a positive control and E. coli 30S subunits as a negative control. Arrows indicate the loading spots (ori), positions of unincorporated [¹⁴C]Phe (mono) as well as the location of di-Phe (di) and penta-Phe peptides (penta) identified on the marker lane (M). Note that peptide products longer than penta-Phe cannot be resolved by this system (indicated by penta+). For quantification the TLC plate was exposed to a phosphoimager screen and analyzed with the Image Quant software. The results of peptide length quantifications using aba2450 (B) or aba2063 (C) ribosomes are shown and compared to native 70S ribosomes as well as to reconstituted wt ribosomes. The produced poly([¹⁴C]Phe) peptides were grouped into the length categories ‘di’ (white), ‘tri+tetra’ (light grey) and ‘penta+’ (dark grey) peptides. Radioactivity values measured in reactions containing no 50S ribosomal subunits [30S in (A)] were subtracted as background values from the corresponding category areas. The total amount of poly([¹⁴C]Phe) detected on each lane was assigned as 1.0. Graphs represent the results of at least two independent in vitro translation experiments, whereas the error bars indicate standard error.

Figure 5.

Natural mRNA in vitro translation is impaired on ribosomes with a disrupted A2450–C2063 base pair. A representative SDS-PAGE of total translation reactions programmed with S8 r-protein mRNA demonstrates full-length [³⁵S]-labeled S8 protein (arrow) produced by reconstituted ribosomes carrying the wt oligo in the PTC. Additional lanes show translation products of ribosomes reconstituted with oligos carrying the abasic site modification (aba) at 2450 or 2063, isoguanosine at 2450 (isoG), purine at 2450 (Pu), or particles reconstituted without synthetic oligo (-oligo). The marker lane (M) shows [³⁵S]-labeled S8 protein produced in vivo in E. coli BL21(DE3) cells. The gel was exposed to a phosphoimager screen and quantified (Image Quant). Background values (radioactivity detected in the areas corresponding to the full-length product in the ‘-oligo’ lanes) were subtracted upon quantification. The asterisk marks a non-specific smear unrelated to translation (see ‘-oligo’ lanes). The quantified relative amounts of the full-length protein (wt was taken as 1.00) is given under the respective lanes.

Figure 6.

Effects of modulating the A2450–C2063 pair on the PTC architecture. (A) In the surface representation of the structure obtained at the end of the MD simulation of the wt PTC, a continuous ‘interaction ring’ is formed involving the A2450–C2063 pair, A2062, a constrained monovalent ion (magenta), the phenylalanine side chain (asterisk) of A-site bound Phe-tRNA^Phe and A76 of deacylated tRNA in the P-site. (B) Removal of the adenine N6-amino group by introducing purine at residue 2450 resulted in the loss of base pairing with C2063 and a complete disconnection of A2062 and the A-site tRNA. (C) Removal of the entire nucleobase at 2450 severely affected the PTC architecture also resulting in the loss of interactions of A2062 with the aa-tRNA in the A-site. Instead the amino acid side chain of Phe-tRNA^Phe seems to interact with the nucleobase at C2063. (D) Removal of the base at 2063 showed less dramatic effects since A2062 was still able to reach the aa-tRNA in the A-site. However, the A2450–C2063 base pair is destroyed as well as A76 of P-tRNA is positioned significantly different in respect to A2450 as compared to the wt situation in (A).