Ribosome Structure, Function, and Early Evolution

Abstract

Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S⁻50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

Keywords: EF-G; EF-Tu; IF2; coevolution; genetic code; ribosome; translation elongation; translation initiation; translocation.

Figure 1

The Shine-Dalgarno-anti-Shine-Dalgarno (SD-ASD) contact in translation initiation on the 30S ribosomal subunit; (A) messenger ribonucleic acid (mRNA, red) with an SD sequence binds the ASD (green) near the 3′-end of the 16S rRNA (PDB 1JGQ). The 16S rRNA is beige (body) and black (head/beak). mRNA lies across the neck. tRNA^fMet (blue) binds in the P-site. Ribosomal proteins are white; (B) detail of the SD-ASD [red-green (carbons)] interaction (PDB 4V4Z).

Figure 2

Formation of a pre-initiation complex on the 30S ribosomal subunit (PDB 5LMV). IF1 (magenta), IF2 (blue), and IF3 (cyan) are present. The fMet-tRNA^fMet (yellow) is bound in the P-site. Other colors are as in Figure 1. The fMet of the P-site fMet-tRNA^fMet bound to IF2 is in the space-filling representation.

Figure 3

Stages of the translation elongation cycle; (A,B) binding of the aa-tRNA·EF-Tu·GTP ternary complex to the A/T site; (C) conformational closing of the 30S subunit and forming the codon-anticodon A-site latch; (D) elbow accommodation of aa-tRNA; (E) full CCA accommodation of aa-tRNA to the A/A-site, release of EF-Tu·GDP, entry of EF-G·GTP; (F) peptidyl transfer; (G) EF-G·GTP→GDP and onset of translocation, opening of the codon-anticodon latch, formation of hybrid tRNA states pe/E and ap/A or ap/ap; (H) full forward and reverse translocation. Some intermediate x-ray or cryo-electron microscopy structures are shown.

Figure 4

EF-Tu and EF-G are homologs that occupy the same site on the ribosome; (A) overlay of ribosome-EF-Tu·GTP (green; PDB 5UYM) and ribosome-EF-G·GDP (red; PDB 4V5M). Overlays were done for ribosomal protein S2. Ribosomes are omitted from the images for simplicity; (B) EF-G·GDP structure (red); (C) EF-Tu·GTP structure (green). At the right, an alignment of Thermus thermophilus EF-G, EF-Tu, and IF2 is shown. Conserved residues (i.e., EF-Tu, Val21, Ile61, and His86) involved in stimulating GTP hydrolysis are indicated. In the alignment, e-values are versus Escherichia coli EF-G.

Figure 5

Closing of the codon-anticodon latch closes the 30S ribosomal subunit; (A) overlay of open and closed latch structures. The head and beak are black (16S: 930–1380). The body is white. tRNA sites locate to the cleft between the head/beak and the body; (B) the latch is open (complex 1); (C) the latch is closed (complex 3). This transition occurs in three stages (Table 1).

Figure 6

50S GTPase-associated complex (GAC) and SRL (ball and stick representation) binding to aa-tRNA·EF-Tu·GTP activates His86 to stimulate GTPase activity. Numbering of EF-Tu residues is as in Figure 4.

Figure 7

Accommodation of aa-tRNA from the A/T-site to the A/A-site. This is a multi-step process that includes elbow accommodation and CCA accommodation (see Figure 3; Table 1); (A) overlay of PDB 5IBB (A/A state) and 5UYM (A/T state); (B) the fully accommodated A/A state poised for peptidyl bond formation; (C) the A/T state before elbow accommodation and CCA accommodation. EF-Tu·GTP is colored magenta.

Figure 8

EF-G·GDP (green) in a compact conformation in a pre-translocation or catalytic state. The compact conformation may be more indicative of an EF-G·GTP catalytic structure. Note the induced kink or bend in the mRNA at the latch that helps position P-site and A-site tRNAs in sufficient proximity for peptidyl transfer.

Figure 9

EF-G GTPase activity and translocation. The tRNA pe/E and ap/A (or ap/ap) hybrid states are observed. The 16S rRNA nucleotides 930–1380 are black (head/beak domain). mRNA (red) occupies the channel between the 16S rRNA body and head/beak (the neck). The codon-anticodon latch is not fully closed and the ap/A tRNA anticodon stem loop (ASL) has disengaged from the latch and has begun to translocate toward the P-site. For the ap/A-site tRNA, the 3′-CCA, where a peptide chain would be attached, makes a typical CCA A-site contact (C75:G2553). The pe/E tRNA 3′-CCA makes a typical E-site contact (U2431, A2432). EF-G·GDP is in an extended conformation supporting forward translocation and acting as a pawl to prevent reverse translocation.

Figure 10

Two thermal rotary motions of the 30S subunit support forward translocation. The 30S subunit reversibly rotates ~7° versus the 50S subunit. The mRNA [red (post translocated) and green (pre-translocated)] threads between the 16S rRNA head/beak domain and the body (the neck), ~18° swiveling of the head/beak domain versus the body drives the mRNA forward and the mRNA:tRNA codon:anticodon attachments into hybrid states. For the pre-translocated state, mRNA and pe/E tRNA are green. 16S rRNA (pre) is white. For the post-translocated state, EF-G·GDP (extended conformation), mRNA, and pe/E site tRNA are yellow, red, and blue. The 16S rRNA (post) is light blue (body) or black (16S head/beak; 930–1380). In the rightmost image, the 16S rRNA body is not shown to simplify the image.

Figure 11

tRNA evolution. Homologous regions have the same color. Ac and T loop stem-loop-stems (17 nt) are red (initially ~CCGGGUUCAAAACCCGG). 5′-As (initially GCGGCGG) and homologous regions are yellow. 3′-As (initially CCGCCGC) and homologous regions are green. The D loop microhelix UAGCC repeat region (originally 17 nt) is magenta. Green spheres are Mg²⁺ ions.

Figure 12

A model for the evolution of abiogenesis, the RNA-protein world, and cellular life. RNA and ribozyme functions that have been generated in vitro are indicated in red. The central advance in evolution of life on earth and biological coding is tRNA. This figure was modified from [64].

Figure 13

Aminoacyl-tRNA synthetases (aaRS) evolution (mostly) in Pyrococcus furiosis (Pfu), an ancient archaea. Columns of the genetic code are indicated by shading: column 1 (red); column 2 (yellow); column 3 (blue); and column 4 (green). Class I and class II aaRS enzymes are indicated with their structural subclasses (A–E). aaRS enzymes with editing active sites are in green type. Boxes are placed around class I aaRS enzymes. Sma: Staphylothermus marinus; Eco: Escherichia coli. AlaX is an editing function missing a synthetic AlaRS active site. This figure is modified from [46].

Figure 14

In archaea, the genetic code is effectively half as complex in tRNA compared to mRNA. The genetic code is shown as a codon-anticodon (Ac) table. The structural classes of aaRS enzymes are indicated (i.e., GlyRS-IIA is indicated as GLY-IIA). Grey shading indicates aaRS editing. Red bases are not utilized in the tRNA anticodon wobble position. Boxes indicate co-evolution of amino acids and aaRS enzymes in columns. TyrRS-IC and TrpRS-IC (boxed) are related across rows. Significantly, only pyrimidine/purine discrimination is achieved in the wobble position in archaea because of anticodon wobble ambiguity. Ancient archaea tend to have ~44 tRNAs, but tRNA wobble U and C anticodons are effectively synonymous.