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Protein biosynthesis takes place in ribosomes – intricately constructed multienzyme complexes in which different rRNAs play an important role (Figure 4.22). rRNAs belong to the most prevalent macromolecules of the cell. The numerous copies of the rDNA cassettes in the genome (Figure 4.21) indicate that this gene must be transcribed very often in order to produce the large number of rRNA molecules that every cell requires. Just for E. coli alone, the number of rRNA molecules is estimated to be 38 000. In a mammalian cell more than 1 million rRNA copies exist. The rRNA genes for 18S, 5.8S, and 26S rRNA are transcribed by RNA polymerase I together, and the individual rRNAs are produced afterward by splicing. Nucleotides of the precursor RNA are chemically modified by snoRNAs before splicing (Figure 2.20).

Figure 4.21 Structure of RNA cassettes and synthesis of rRNA. ITSs, internal transcribed spacers; IGSs, intergenic spacers; 5S rRNA genes are transcribed separately.

Figure 4.22 shows the assembled building blocks of prokaryotic and eukaryotic ribosomes. As mitochondria and chloroplasts contain their own ribosomes, which originated from bacteria (see Section 3.1.3), the expected type of rRNAs corresponds to those of bacteria (note that in mitochondria a 12S rRNA is present instead of the 23S rRNA).

Figure 4.22 Structure of (a) prokaryotic and (b) eukaryotic ribosomes. For the structure of rRNA, see Figure 2.20.

16/18S rRNA and 23/28S rRNAs exhibit complex spatial structures, which are conserved over a wide range of organisms (Figure 2.20). Even though the RNAs consist of single strands, they form complementary double strands (so‐called stem structures) at many sites in aqueous environments. The nucleotide sequence of stem structures is very strongly preserved in evolution. The situation is different for the loops, in which the nucleotides have been modified posttranscriptionally. This phenomena of base modification is especially observed with tRNAs (but also in rRNAs), in which more than 50 modified nucleotides have been discovered. Substituted bases are thiouracil, 5‐methylcytosine, dihydrouracil, thiothymine, thiocytosine, N⁴‐acetylcytosine, 1‐methylhypoxanthine, 1‐methylguanine, and N⁶‐methyladenine. There are comparatively many substitutions, deletions, insertions, and inversions present in the loops. Before NGS, genetic trees of all organisms have been reconstructed from the nucleotide sequences of the conserved rRNAs, giving them a special role in molecular evolution. The tree of life and the classification of species were largely based on the analysis of conserved rDNA genes. Today, because of the wide availability of NGS, such trees are often reconstructed from partial genomes (see Chapter 1).

The ribosomal proteins are arranged around the rRNA, together constituting a complex nanomachine known as the ribosome (Figures 4.22 and 4.23). Both ribosomal subunits are assembled in the cell nucleolus and are transported individually into the cytosol through the nuclear pores. Free mRNA molecules are recognized by the small subunits, which are first loaded with methionine tRNA and guanosine triphosphate (GTP)‐activated initiation factors (eIF‐2). The small subunit slides along the mRNA until the first start codon AUG is reached, where methionine tRNA is bound via its anticodon UTC. Following the dissociation of the initiation factor eIF‐2, the large ribosomal subunit is able to bind, and the ribosome is positioned ready to begin translation. There are three formally distinguished binding sites: the arriving aminoacyl‐tRNAs bind to the A‐site, the tRNA with the peptide chain sits in the P‐site, and the E‐site releases the free tRNA after peptide transfer (Figure 4.23).

Figure 4.23 Schematic illustration of protein biosynthesis in ribosomes. Three binding sites are distinguished in ribosomes: E, P, and A.

In the A‐site, the arriving aminoacyl‐tRNAs (loaded with amino acids) are hybridized via their anticodon to the corresponding triplet codon on the mRNA (Figure 4.24). In the next step, the peptide residue on the tRNA in the P‐site is transferred to the aminoacyl‐tRNA in the A‐site (peptidyl transfer is catalyzed by the rRNA; Figure 4.25). Next, the ribosome moves along three nucleotides on the mRNA and releases the free tRNA from the P‐site, which now carries the tRNA with the growing peptidyl residue. These steps are repeated until a stop codon is reached. A specific release factor then binds and blocks access for further aminoacyl‐tRNAs to the A‐site. As a consequence, the peptide chain is released.

Figure 4.24 Loading tRNA with an amino acid. First the amino acid is activated through the binding of ATP. The activated amino acid is transferred to the 3′‐OH group of the terminal adenine residue of the tRNA, and an adenosine monophosphate (AMP) residue is set free. This reaction is catalyzed by aminoacyl‐tRNA synthetase that is specific for every amino acid. aa‐tRNA, aminoacyl‐tRNA (i.e. a tRNA loaded with an amino acid).

Figure 4.25 rRNA‐catalyzed peptide transfer in ribosomes. (a) Possible reaction mechanism with an adenine residue of the rRNA participating in catalysis. (b) Reaction pathway of peptidyl transfer.

After protein synthesis, the newly synthesized proteins fold themselves into the correct conformation, aided in many cases by chaperones (e.g. diverse heat shock proteins; hsp60 and hsp70 and others) acting as auxiliary enzymes. Incorrectly folded or incorrectly synthesized proteins (e.g. protein fragments resulting from strand breaking) are coupled with the protein ubiquitin and are broken down in a cellular “shredder” – the proteasomes.

Protein biosynthesis can occur on free ribosomes in the cytoplasm or on ribosomes, which bind to the rough ER (see Chapter 5). A single mRNA can be used by several ribosomes concomitantly; such structures are called polyribosome.

Prokaryoticand eukaryotic ribosomes are constructed according to a very similar pattern (Figure 4.22), and protein biosynthesis is conducted according to very similar principles. However, the particular rRNAs and ribosomal enzymes exhibit important differences. The importance of many antibiotics depends on these differences to specifically inhibit prokaryotic ribosomes. Many antibiotics intervene in bacterial protein biosynthesis (Table 4.7).

Table 4.7 Protein biosynthesis in bacterial ribosomes as a target for antibiotics.

Antibiotic	Mode of action
Tetracycline	Inhibits A‐site in ribosomes
Aminoglycosides (streptomycin)	Disturbs anticodon–codon recognition and chain elongation
Erythromycin	Binds to 50S subunit, blocks exit site (E), and inhibits chain elongation
Chloramphenicol	Binds to 50S subunit and inhibits peptidyl transfer
Puromycin	Induces a premature chain termination

Owing to their selectivity toward bacteria, antibiotics (which came on the market only 70 years ago) are generally substances with few side effects in humans. The search for new and more effective antibiotics is still one of the most important challenges of biotechnology and medicine because many pathogens have become resistant (overexpression of ABC transporters, target site mutations) to existing antibiotics (multidrug‐resistant(MDR) pathogens). A number of pathogenic strains of Staphylococcus aureus that have become resistant to most antibiotics (so‐called methicillin‐resistant S. aureus [MRSA]) are particularly dangerous (see Section 3.2).