Genes VII

Ribosomes are ribonucleoprotein particles in which a majority of the mass is provided by rRNA. The shapes of all ribosomes are generally similar, but only those of bacteria (70S) have been characterized in detail. The small (30S) subunit has a squashed shape, with a "body" containing about two-thirds of the mass divided from the "head" by a cleft. The large (50S) subunit is more spherical, with a prominent "stalk" on the right and a "central protuberance." Locations of all proteins are known approximately in the small subunit.

Each subunit contains a single major rRNA, 16S and 23S in prokaryotes, 18S and 28S in eukaryotic cytosol. There are also minor rRNAs, most notably 5S rRNA in the large subunit. Both major rRNAs have extensive base pairing, mostly in the form of short, imperfectly paired duplex stems with single-stranded loops. Conserved features in the rRNA can be identified by comparing sequences and the secondary structures that can be drawn for rRNA of a variety of organisms. The 16S rRNA has four distinct domains; the three major domains have been mapped into regions of the small subunit. Eukaryotic 18S rRNA has additional domains. One end of the 30S subunit may consist largely or entirely of rRNA.

Each subunit has several active centers, concentrated in the translational domain of the ribosome where proteins are synthesized. Proteins leave the ribosome through the exit domain, which can associate with a membrane. The major active sites are the P and A sites, the E site, the EF-Tu and EF-G binding sites, peptidyl transferase, and mRNA-binding site. Ribosomal proteins required for the function of some of these sites have been identified, but the sites have yet to be mapped in terms of three-dimensional ribosome structure. Ribosome conformation may change at stages during protein synthesis; differences in the accessibility of particular regions of the major rRNAs have been detected.

The major rRNAs contain regions that are localized at some of these sites, most notably the mRNA-binding site and P site on the 30S subunit. The 3′ terminal region of the rRNA seems to be of particular importance. Functional involvement of the rRNA in ribosomal sites is best established for the mRNA-binding site, where mutations in 16S rRNA affect the initiation reaction. Ribosomal RNA is also the target for some antibiotics or other agents that inhibit protein synthesis. 23S rRNA appears to possess the essential catalytic activity of peptidyl transferase.

A codon in mRNA is recognized by an aminoacyl-tRNA, which has an anticodon complementary to the codon and carries the amino acid corresponding to the codon. A special initiator tRNA (fMet-tRNA_f in prokaryotes or Met-tRNA_i in eukaryotes) recognizes the AUG codon, which is used to start all coding sequences. In prokaryotes, GUG is also used. Only the termination (nonsense) codons UAA, UAG, and UGA are not recognized by aminoacyl-tRNAs.

Ribosomes are released from protein synthesis to enter a pool of free ribosomes that are in equilibrium with separate small and large subunits. Small subunits bind to mRNA and then are joined by large subunits to generate an intact ribosome that undertakes protein synthesis. Recognition of a prokaryotic initiation site involves binding of a sequence at the 3′ end of rRNA to the Shine-Dalgarno motif which precedes the AUG (or GUG) codon in the mRNA. Recognition of a eukaryotic mRNA involves binding to the 5′ cap; the small subunit then migrates to the initiation site by scanning for AUG codons. When it recognizes an appropriate AUG codon (usually but not always the first it encounters) it is joined by a large subunit.

A ribosome can carry two aminoacyl-tRNAs simultaneously: its P site is occupied by a polypeptidyl-tRNA, which carries the polypeptide chain synthesized so far, while the A site is used for entry by an aminoacyl-tRNA carrying the next amino acid to be added to the chain. The polypeptide chain in the P site is transferred to the aminoacyl-tRNA in the A site and then the ribosome translocates one codon along the mRNA. Translocation and several other stages of ribosome function require hydrolysis of GTP.

Protein synthesis is an expensive process. ATP is used to provide energy at several stages, including the charging of tRNA with its amino acid, and the unwinding of mRNA. It has been estimated that up to 90% of all the ATP molecules synthesized in a rapidly growing bacterium are consumed in assembling amino acids into protein!

Additional factors are required at each stage of protein synthesis. They are defined by their cyclic association with, and dissociation from, the ribosome. IF factors are involved in prokaryotic initiation. IF-3 is needed for 30S subunits to bind to mRNA and also is responsible for maintaining the 30S subunit in a free form. IF-2 is needed for fMet-tRNA_f to bind to the 30S subunit and is responsible for excluding other aminoacyl-tRNAs from the initiation reaction. GTP is hydrolyzed after the initiator tRNA has been bound to the initiation complex. The initiation factors must be released in order to allow a large subunit to join the initiation complex.

Prokaryotic EF factors are involved in elongation. EF-Tu binds aminoacyl-tRNA to the 70S ribosome. GTP is hydrolyzed when EF-Tu is released, and EF-Ts is required to regenerate the active form of EF-Tu. EF-G is required for translocation. Binding of the EF-Tu and EF-G factors to ribosomes is mutually exclusive, which ensures that each step must be completed before the next can be started. RF factors are required for termination. Protein synthesis in eukaryotes is generally similar to the process in prokaryotes, but involves a more complex set of accessory factors.