Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation

Abstract

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure. The mass center of gravity of the (tRNA)2mRNA complex moves within the ribosome by 12 +/- 4 A in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110 degrees +/- 10 degrees before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18 degrees.

Figure 1

The basic scattering functions [UH] for the pre- (A) and post-translocational (B) states (relative intensities versus the momentum transfer, Q). The cross-term [UH] can be measured with the highest precision from the three basic scattering functions (7), and thus gives the most solid information about the localization of the label within the 70S ribosome. The line indicates the fit to the experimental data points by using the 70S model of the Frank group (11) and the eight-sphere model of the tRNAs (Fig. 2).

Figure 2

Eight-sphere model of two tRNAs. The dots indicate the phosphorus atoms from the crystal structure of tRNA^Phe (dark blue, CCA-3′ ends; red, anticodons; refs. and 16). The tRNAs are shown in opposite arrangement (ψ = 180°).

Figure 3

Orientation of the tRNAs within the ribosome for the pre- (A and B) and the post- translocational (C) states. The orientations of the tRNAs are depicted by lines passing through the midpoint between the two anticodons and that between the two CCA ends. (A) Orientations with Δ values of 10% above the minimal deviation (1.479, calculated according to Eq. 1). (B and C) Orientations with Δ values of 5% above the minimal deviation (1.479 and 1.334, respectively).

Figure 4

Mutual arrangement of the tRNAs in the pre- and the post-translocational states. (A) Schematic representation of the R configuration in PRE by using the angle of 110°. (B) Dependence of the deviation Δ (Eq. 1) on the angle ψ between both tRNAs. The left half represents the R configuration, and the right half represents the S configuration (see also Insert). The minimal deviation is found at angles of ψ = 110° ± 10°. (C) Schematic representation of the S configuration in PRE by using the angle of 110°.

Figure 5

tRNA arrangement in the PRE (Left) and POST states (Right). The green and red tRNAs are thought to be at the A and P sites, respectively, of PRE states and at the P and E sites, respectively, of POST states. Only the R configuration is shown. (A and B) 70S ribosome. (C and D) 30S subunit. (E and F) 50S subunit.