Protein synthesis by single ribosomes

Abstract

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

FIGURE 1.

AFM images of ribosomes tightly adsorbed to freshly cleaved mica. The concentrations of ribosomes applied in binding buffer are shown.

FIGURE 2.

Poly(Phe) synthesis as measured by incorporation of [³H]Phe in a TCA-precipitable peptide. (A) Synthesis by ribosomes adsorbed on mica (squares: in the presence of elongation factors Tu, G, and Ts; diamonds: in the absence of elongation factors). (B) Synthesis by ribosomes in solution. The values shown are the mean and standard deviation (n = 4).

FIGURE 3.

Principle of the tethered particle method. (A) A microsphere (shown in pink) is tethered at the 3′ end of an mRNA molecule, which is bound to a ribosome immobilized on a microscope slide. The mRNA molecule is shown as a curvy black line, the end-to-end length of the tether is shown as a black dotted line, and the peptide synthesized by the ribosome is shown in orange. The microsphere can diffuse only within the range limited by the surface of the slide and the length of the tether, as shown by the green dashed line and shading. (B) As peptide synthesis proceeds, the ribosome pulls the 3′ end of the mRNA toward itself, reducing the range of restricted diffusion (schematic not drawn to scale).

FIGURE 4.

Restricted diffusion. (A) Centroid distributions of a bead displaying restricted diffusion. The flow chamber contained all the constituents necessary to support poly(U) programmed poly(Phe) synthesis by the ribosomes immobilized on the surface. The distribution was measured at 17, 54, and 95 min after sealing the chamber, as indicated by the magenta, orange, and black symbols, respectively. (B) For the same bead, D_rms is plotted as a function of time (green circles); D_rms of an immobile bead present in the same microscopic field is also plotted (red squares), as well as the D_rms of a bead in a different field, displaying a large range of diffusion throughout the time of the experiment (blue triangles). (C) TPM data pooled from six beads displaying a reduction of D_rms with time. The recordings from different beads were aligned by shifting each set of data to the origin at the first measurement. Each set of data is represented by a different symbol. The line is a linear regression through the pooled data, yielding a slope of −2.8 ± 0.3 (standard error) nm/min; r² = 0.73.