New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Abstract

Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.

FIGURE 1.

Synthetic schemes and structures of photoreactive nucleotides and modified tRNA ligands. (A) Synthesis of 2-azido-2′-O-methyladenosine 3′,5′-bisphosphate (p2N₃2′OMeAp). (B) Synthesis of 2-azido-2′-deoxyadenosine 3′,5′-bisphosphate (p2N₃dAp) and its final incorporation into the photoreactive transition-state analog (2N₃dA76)tRNA^Phe-p-Puro.

FIGURE 2.

Analysis of modified photoreactive tRNA ligands. (2N₃A76)tRNA^Phe, (2N₃dA76)tRNA^Phe, (2N₃2′OMeA76)tRNA^Phe, (2N₃dA76)tRNA^Phe-p-Puro, (2N₃dA76)tRNA^Phe-p, and unmodified tRNA^Phe, labeled with ³²P as described in Materials and Methods, were subjected to digestion with RNase T1 under partial conditions to avoid overdigestion. In addition, (2N₃dA76)]tRNA^Phe-p-Puro and (2N₃dA76)tRNA^Phe-p were subjected to partial acid hydrolysis (60 min in 1% trifluoroacetic acid at 23°C) after T1 digestion. Partial alkaline digestion of (2N₃dA76)tRNA^Phe-p was used to generate a reference ladder. The samples were resolved by denaturing 25% PAGE and visualized by autoradiography. Labeled products released from the 3′ ends of the photoreactive tRNA ligands are CACC(2N₃dA), CACC(2N₃2′OMeA), and CACC(2N₃A) (collectively designated as CACC[2N₃A*] in the figure), CACC(2N₃dA)-p, and CACC(2N₃dA)-p-Puro. A characteristic mobility shift of about 6 nt in the 3′ T1 RNase product is observed when (2N₃dA76)tRNA^Phe-p is condensed with puromycin. The observed 6-nt shift in (2N₃dA76)tRNA^Phe-p-Puro is reversed upon treatment with acid, consistent with hydrolysis of phosphoramidate linkage between (2N₃dA76)tRNA^Phe-p and puromycin (Benkovic and Sampson 1971; Welch et al. 1995).

FIGURE 3.

Competition binding assays. Radiolabeled (A) unmodified tRNA^Phe, (B) (2N₃A76)tRNA^Phe, (C) (2N₃2′OMeA76)tRNA^Phe, (D) (2N₃dA76)tRNA^Phe, and (E) (2N₃dA76)tRNA^Phe-p-Puro (∼100 nM) were first mixed with various concentrations of unlabeled, unmodified tRNA^Phe and then mixed with 70S ribosomes (10 nM) in the presence of polyU and 20 mM MgCl₂. The binding reactions were incubated at 4°C for 48 h to reach equilibrium and then analyzed by the nitrocellulose filter binding assay. Error bars represent the standard deviation of triplicate samples of each reaction. The concentrations of competing unlabeled, unmodified tRNA^Phe that displace 50% of the specifically bound radiolabeled ligand (IC₅₀) were calculated using the nonlinear regression function of GraphPad Prism software. In these assays, radiolabeled ligands with higher affinities for the ribosome lead to higher IC₅₀ values.

FIGURE 4.

Sucrose-gradient profiles of dissociated ribosomal subunits after cross-linking. (A) unmodified tRNA^Phe, (B) (2N₃A76)tRNA^Phe, (C) (2N₃dA76)tRNA^Phe, (D) (2N₃2′OMeA76)tRNA^Phe, and (E) (2N₃dA76)]tRNA^Phe-p-Puro. Both UV absorbance (solid line) and radioactivity (dashed line with data points) were monitored as the gradients were fractionated. With the exception of unmodified tRNA^Phe, the ligands cross-linked exclusively to the 50S subunit, as shown by the comigration of radioactivity with the 50S peak.

FIGURE 5.

Analysis of the distribution of cross-links between 23S rRNA and 50S-subunit proteins. (A) ³²P-labeled material isolated from 50S subunits was resuspended in 6 M urea, resolved by 4%–20% gradient SDS-PAGE, and visualized by autoradiography. With the exception of unmodified tRNA^Phe, the ligands cross-linked to both 23S rRNA and to proteins. (B) Bar graph showing the relative distribution of cross-linked 23S rRNA and protein estimated by PhosphorImager analysis of the gels. Error bars represent the standard deviation in measurements obtained from three separate SDS-PAGE runs.

FIGURE 6.

Identification of ribosomal proteins cross-linked by each photoreactive ligand. 50S-subunit components were solubilized in 6 M urea and incubated with ribonuclease S1, which entirely digested the RNA, leaving a radioactively labeled 5′-phosphate and its modified A76 nucleotide covalently attached to a ribosomal protein. The digested material was resolved by (A) 10% SDS-PAGE and by (B) two-dimensional PAGE, and then visualized by autoradiography. In panel A the labeled bands contain different derivatives of A76, collectively designated as A*. In panel B, the samples were spiked with nonradioactive proteins that were acid-extracted from purified 70S ribosomes to provide reference markers. The proteins in the two-dimensional (2D) gel were stained with bromophenol blue and the image was overlaid with the autoradiogram of the same gel. The positions of (p2NA)-L27, (p2NA)-L33, and (2NdA-p-Puro)-L27 are displaced from those of L27 and L33 owing to the presence of the negatively charged nucleotide moieties (Maguire et al. 2005).