The Ribosome Ribosomes a re the sites of protein synthesis in both prokaryotic and eukaryotic cells.

First characterized as particles detected by ultracentrifugation of celllysates, ribosomes are usually

designated according to their rates of sedimentation: 70S for bacterial ribosomes and 80S for the
somewhat larger ribosomes of eukaryotic cells. Both prokaryotic and eukaryotic ribosomes are
composed of two distinct subunits, each containing characteristic proteins and rRNAs. The fact that cells
typically contain many ribosomes reflects the central importance of p rotein synthesis in cell
metabolism. E. coli, for example, contain about 20,000 ribosomes, which acco unt for approximately
25% of the dry weight of the cell, and rapidly growing mammalian cells contain about 10 million
ribosomes.

The general structures of prokaryotic and eukaryotic ribosomes are similar, although they differ in some
details (Figure 8.4). The small subunit (designated 305) of E. coli ribosomes consists of the 165 rRNA and
21 proteins; the large subunit (50S) is composed of the 235 and 55 rRNAs and 34 proteins. Each
ribosome contains one copy of the rRNAs and one copy of each of the ribosomal proteins, with one
exception: One protein of the 50S subunit is present in four copies. The subunits of eukaryotic
ribosomes are larger and contain more proteins than their prokaryotic counterparts. The small subunit
(405) of eukaryotic ribosomes is composed of the 185 rRNA and approximately 30 proteins; the large
subunit (60S) contains the 285, 5.85, and 55 rRNAs and about 45 proteins. Because of their large size
and complexity, high-resolution structural analysis of ribosomes by X-ray crystallography was not
accomplished until 2000, when structures of both the 50S and 305 subunits were first reported. As
discussed below, understanding the structure of ribosomes at the atomic level has had a major impact
on our understanding of ribosome function. A noteworthy feature of ribosomes is that they can be
formed in vitro by self-assembly of their RNA and protein constituents. As first described in 1968 by
Masayasu Nomura, purified ribosomal proteins and rRNAs can be mixed together and, under
appropriate conditions, will re-form a functional ribosome. Although ribosome assembly in vivo
(particularly in eukaryotic cells) is considerably more complicated, the ability of ribosomes to
selfassemble in vitro has provided an important experimental tool, a llowing analysis of the roles of
individual proteins and rRNAs. Like tRNAs, rRNAs form characteristic secondary structures by
complementary base pairing (Figure 8.5). In association with ribosomal proteins, the rRNAs fold further,
into distinct three-dimensional structures. Initially, rRNAs were thought to play a structural role,
providing a scaffold upon which ribosomal proteins assemble. However, with the discovery of the
catalytic activity of other RNA molecules (e.g., RNase P and the self-splicing introns discussed in Chapter
7), the possible catalytic role of rRNA became widely considered. Consistent with this hypothesis, rRNAs
were found to be absolutely required for the in vitro assembly of functional ribosomes. On the other
hand, the omission of many ribosomal proteins resulted in a decrease, but not a complete loss, of
ribosomal activity. Direct evidence for the catalytic activity of rRNA first came from experiments of Harry
Noller and his colleagues in 1992. These investigators demonstrated that the large ribosomal subunit is
able to catalyze the formation of peptide bonds (the peptidyl transferase reaction) even after
approximately 90% of the ribosomal proteins have been removed by standard protein extraction
procedures. In contrast, treatment with RNase completely abolishes peptide bond formation, providing
strong support for the hypothesis that the formation of a peptide bond is an RNA-catalyzed reaction.
However, some of the ribosomal proteins could not be removed under conditions that left the ribosomal
RNA intact, so the role of ribosomal proteins as catalysts of peptide bond formation could not be
definitively ruled out. Unambiguous evidence that protein synthesis is catalyzed by rRNA came from the
first high-resolution stmctural analysis of the 50S ribosomal subunit, which was reported by Peter
Moore, Thomas Seitz, and their colleagues in 2000 (Figure 8.6). This atomic-level view of ribosome
structure revealed that ribosomal proteins were strikingly absent from the site at which the peptidyl
transferase reaction occurred, making it evident that rRNA was responsible for catalyzing peptide bond
formation. The large ribosomal subunit thus functions as a ribozyme, with the fundamental reaction of
protein synthesis being catalyzed by ribosomal RNA. Rather than being the primary catalytic
constituents of ribosomes, ribosomal proteins are now thought to play a largely structural role. The
direct involvement of rRNA in the peptidyl transferase reaction has important evolutionary implications.
RNAs are thought to have been the first self-replicating macromolecules (see Chapter 1). This notion is
strongly supported by the fact that ribozymes, such as RNase P and self-splicing introns, can catalyze
reactions that involve RNA substrates. The role of rRNA in the formation of peptide bonds extends the
catalytic activities of RNA beyond self-replication to direct involvement in protein synthesis. Additional
studies indicate that the Tetrahymena rRNA ribozyme can catalyze the attachment of amino acids to
RNA, lending credence to the possibility that the original aminoacyl tRNA synthetases were RNAs rather
than proteins. The ability of RNA molecules to catalyze the reactions required for protein synthesis as
well as for self-replication may provide an important link for understanding the early evolution of cells.