smFRET study of rRNA dimerization at the peptidyl transfer center

Abstract

The ribosome is a ribozyme. At the peptidyl transfer center (PTC) of 180 nt, two loops (the A- and P- loops) bind to tRNAs and position them in close proximity for efficient peptidyl ligation. There is also a 2-fold rotational symmetry in the PTC, which suggests that the precursor of the modern ribosome possibly emerged through dimerization and gene fusion. However, experiments that demonstrate the possible dimerization have not yet been published. In our investigation, we reported single molecule FRET studies of two RNA fragments that generated high FRET values. By dye-labeling the 5'-biotinylated rRNA molecules at the 3'- terminals, or labeling three different types of tRNA-like oligos, we observed that RNA scaffolds can assemble and bring several short tRNA-acceptor-domain analogs, but not full-length tRNAs, to close proximity. Mg²⁺ and continuous 3-way junction motifs are essential to this process, but amino acid charging to the tRNA analogs is not required. We observed RNA dimers via native gel-shifting experiments. These experiments support the possible existence of a proto-ribosome in the form of an RNA dimer or multimer.

Keywords: Gel shifting; LUCA (last universal common ancestor); Peptide ligase; Peptidyl transferase activity; Single molecule FRET; tRNA-like oligo.

Figure 1.

(A) Illustration of the RNA/DNA sequences relative to the canonical ribosomal peptidyl transfer center. The tip of H89 is truncated and replaced with GUGA tetraloop. (B) RNA dimerization interactions between PTC1a (green) and PTC1b (red). The e coli numbers of the bases are labelled in Figure S1.

Figure 2.

Illustration of the smFRET setup and a representative FRET image. The fluorescence emissions from Cy3 and Cy5 were collected by cameras 2 and 1, respectively, and overlaid with colors.

Figure 3.

FRET efficiency histograms between the PTC_DNA. The green and red stars showed the Cy3/Cy5 labeling positions. The second and third columns showed the experimental data with 5 and 15 mM Mg²⁺, respectively. The FRET efficiency peaks were labelled in the plots.

Figure 4.

Agarose native gel (stained with SYBR^™Gold) and FRET measurement of RNA dimers. (A) Lane 1: single strand RNA ladder; Lane 2 and 3: PTC1b (100 and 200 ng); Lane 4: PTC1a (200 ng); Lane 5: PTC1a/b (100ng/100ng). (B) FRET efficiency histograms between the RNA molecules. The green and red starts showed the Cy3 and Cy5 labeling positions, respectively. (C) and (D) Similar FRET efficiency histograms were observed with 5 and 15 mM Mg²⁺.

Figure 5.

FRET efficiency histograms between the RNA-RNA scaffolds. The first column showed the FRET between PTC1a/1b. The second column showed the FRET between PTC 1b/1b. Three Mg²⁺ concentrations were studied.

Figure 6.

FRET efficiency histograms between tRNA-like oligos at 10 mM of Mg²⁺. (A) Relative position of Cy3 (green star)/Cy5 (red star) labelled oligos. (B) 30% denaturing PAGE Gel assay of Cy5 labelled oligos. Lane 1: CA_DNA; Lane 2: ACCCACCA; Lane 3: Lys-tRNALys_T1. (C-E) FRET of uncharged CA_DNA ; charged Lys-tRNALys_T1; charged Lysine-minihelix.