Evolved orthogonal ribosome purification for in vitro characterization

Abstract

We developed orthogonal ribosome-mRNA pairs in which the orthogonal ribosome (O-ribosome) specifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, they have been used to provide new information about the ribosome, and they form a crucial part of foundational technologies for genetic code expansion and encoded and evolvable polymer synthesis in cells. The production of O-ribosomes in the cell makes it challenging to study the properties of O-ribosomes in vitro, because no method exists to purify functional O-ribosomes from cellular ribosomes and other cellular components. Here we present a method for the affinity purification of O-ribosomes, via tagging of the orthogonal 16S ribosomal RNA. We demonstrate that the purified O-ribosomes are pure by primer extension assays, and structurally homogenous by gel electrophoresis and sucrose gradients. We demonstrate the utility of this purification method by providing a preliminary in vitro characterization of Ribo-X, an O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to amber codons. Our data suggest that the basis of Ribo-X's in vivo activity is a decreased affinity for RF1.

Figure 1.

Design of the ribosome RNA affinity tag. The structure of the 70S ribosome (21) showing the position of the tag (shown in red) with the 30S shown in yellow and the 50S in grey. The position and sequence of the tag in the 16S secondary sequence is shown below. The blue nucleotides are the linker sequence and the affinity tag is shown in green (12).

Figure 2.

O-ribosome affinity purification strategy. Initial binding of the tagged O-ribosome to glutathione bound GST-MS2 is followed by elution using the RNA tag sequence.

Figure 3.

Characterization of affinity purified tagged O-ribosomes. (a) Purity of eluted O-ribosome measured by allele-specific primer extension. (b) Analytical sucrose gradient profiles of untagged wild-type and purified tagged O-ribosome. (c) Non-denaturing gel separation the indicated ribosomes.

Figure 4.

In vitro termination assays reveal that Ribo-X has a decreased affinity for RF1. (a) Schematic of termination assays. A complex of the O-ribosome with an initiator tRNA aminoacylated with ³⁵S formyl-methionine (red circle) is assembled (Step 1), in subsequent rounds this requires dissociation of deacetylated tRNA. RF1 is added (Step 2) directing hydrolysis of ³⁵S formyl-methionine from the tRNA (Step 3). RF1 dissociates (Step 4). The filter-binding assay measures the ribosome associated ³⁵S, while the TCA precipitation measures the ³⁵S released (b) RF1 termination activity measured in vitro with untagged wild-type and purified tagged O-ribosome, using filter-binding assays. (c) O-ribosome and Ribo-X termination activity with RF1. Measurement of initial rate of tRNA-fMet hydrolysis over a range of RF1 concentrations is shown for O-ribosome and Ribo-X. RF1 concentration was increased till there was a measurable release in fMet and this release plotted. tRNA-fMet hydrolysis (37°C, 10 s) is measured by TCA precipitation.