Protein Synthesis:- The Ribosome Is a Complex Supramolecular Machine
Each E. coli cell contains 15,000 or more ribosomes, making up almost a quarter of the dry weight of the cell. Bacterial ribosomes contain about 65% rRNA and 35% protein; they have a diameter of about 18 nm and are composed of two unequal subunits with sedimentation coefficients of 30S and 50S and a combined sedimentation coefficient of 70S. Both subunits contain dozens of ribosomal proteins and at least one large rRNA (Table 27–6). Following Zamecnik’s discovery that ribosomes are the complexes responsible for protein synthesis, and following elucidation of the genetic code, the study of ribosomes accelerated. In the late 1960s Masayasu No mura and colleagues demonstrated that both ribosomal subunits can be broken down into their RNA and protein components, then reconstituted in vitro. Under ap propriate experimental conditions, the RNA and protein spontaneously reassemble to form 30S or 50S subunits nearly identical in structure and activity to native sub units. This breakthrough fueled decades of research into the function and structure of ribosomal RNAs and proteins. At the same time, increasingly sophisticated structural methods revealed more and more details about ribosome structure. The dawn of a new millennium brought with it the elucidation of the first high-resolution structures of bacterial ribosomal subunits. The bacterial ribosome is com plex, with a combined molecular weight of 2.7 million, and it is providing a wealth of surprises (Fig. 1). First, the traditional focus on the protein components of ribosomes was shifted. The ribosomal subunits are huge RNA molecules. In the 50S subunit, the 5S and 23S rRNAs form the structural core. The proteins are secondary elements in the complex, decorating the surface. Second and most important, there is no protein within 18 Å of the active site for peptide bond formation. The high-resolution structure thus confirms what many had suspected for more than a decade: the ribosome is a ribozyme. In addition to the insight they provide into the mechanism of protein synthesis (as elaborated below), the detailed structures of the ribosome and its subunits have stimulated a new look at the evolution of life (Box 27–3). The two irregularly shaped ribosomal subunits fit together to form a cleft through which the mRNA passes as the ribosome moves along it during translation (Fig. 1b). The 55 proteins in bacterial ribosomes vary enormously in size and structure. Molecular weights range from about 6,000 to 75,000. Most of the proteins have globular domains arranged on the ribosome sur face. Some also have snakelike protein extensions that protrude into the rRNA core of the ribosome, stabilizing its structure. The functions of some of these proteins have not yet been elucidated in detail, although a structural role seems evident for many of them. The sequences of the rRNAs of many organisms are now known. Each of the three single-stranded rRNAs of E. coli has a specific three-dimensional conformation featuring extensive intrachain base pairing. The predicted secondary structure of the rRNAs (Fig. 2) has largely been confirmed in the high-resolution models, but fails to convey the extensive network of tertiary in teractions evident in the complete structure.
FIGURE 1 Ribosomes. Our understanding of ribosome structure took a giant step forward with the publication in 2000 of the high-resolution structure of the 50S ribosomal subunit of the bacterium Haloarcula marismortui by Thomas Steitz, Peter Moore, and their colleagues. This was followed by additional high-resolution structures of the ribosomal subunits from several different bacterial species, and models of the corresponding complete ribosomes. A sampling of that progress is presented here. (a) The 50S and 30S bacterial subunits, split apart to visualize the surfaces that interact in the active ribosome. The structure on the left is the 50S subunit (derived from PDB ID 1JJ2 and 1GIY), with tRNAs (purple, mauve, and gray); bound to sites E, P, and A, described later in the text; the tRNA anti codons are in orange. Proteins appear as blue wormlike structures; the rRNA as a blended space-filling representation designed to highlight surface features, with the bases in white and the backbone in green. The structure on the right is the 30S subunit (derived from PDB ID 1J5E and 1JGO). Proteins are yellow and the rRNA white. The part of the mRNA that interacts with the tRNA anti codons is shown in red. The rest of the mRNA winds through grooves or channels on the 30S subunit surface. (b) A model of a complete active bacterial ribosome (derived from PDB ID 1J5E, 1JJ2, 1JGO, and 1GIY). All components are colored as in (a). This is a view down into the groove separating the sub units. A second view (inset) is from the same angle, but with the tRNAs removed to give a better sense of the cleft where protein synthesis occurs.
(c) Structure of the 50S bacterial ribosome subunit (PDB ID 1Q7Y). The subunit is again viewed from the side that attaches to the 30S sub unit, but is tilted down slightly compared to its orientation in (a). The active site for peptide bond formation (the peptidyl transferase activity), deep within a surface groove and far away from any protein, is marked by a bound inhibitor, puromycin (red). (d) Summary of the composition and mass of ribosomes in prokaryotes and eukaryotes. Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge. The S values are not necessarily additive when subunits are combined, because rates of sedimentation are affected by shape as well as mass.
The ribosomes of eukaryotic cells (other than mitochondrial and chloroplast ribosomes) are larger and more complex than bacterial ribosomes (Fig. 27–9d), with a diameter of about 23 nm and a sedimentation co efficient of about 80S. They also have two subunits, which vary in size among species but on average are 60S Yeast alanine tRNA (tRNAAla), the first nucleic acid to be completely sequenced (Fig. 27–11), contains 76 nucleotide residues, 10 of which have modified bases. Comparisons of tRNAs from various species have revealed many common denominators of structure (Fig. 27–12). Eight or more of the nucleotide residues have modified bases and sugars, many of which are methylated derivatives of the principal bases. Most tRNAs have a guanylate (pG) residue at the 5end, and all have the trinucleotide sequence CCA (3) at the 3end. When and 40S. Altogether, eukaryotic ribosomes contain more than 80 different proteins. The ribosomes of mitochondria and chloroplasts are somewhat smaller and simpler than bacterial ribosomes. Nevertheless, ribosomal structure and function are strikingly similar in all organisms and organelles.
FIGURE 2 Bacterial rRNAs. Diagrams of the secondary structure of E. coli 16S and 5S rRNAs. The first (5 end) and final (3 end) ribonucleotide residues of the 16S rRNA are numbered.
