Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
occurs in a number of ways. One way is methylation of a specific adenine base in the 23S rRNA, which prevents binding of the antibiotic, by enzymes called the Erm methylases. Some mutational changes in the 23S rRNA can also confer resistance to these antibiotics. Resistance can also occur by acquisition of genes that encode efflux pumps that remove the antibiotic from the cell. New derivatives of the antibiotics must be made constantly to stay ahead of the advancing bacterial resistance.
Thiostrepton
Thiostrepton and other thiopeptide antibiotics block translation by binding to 23S rRNA in the region of the ribosome involved in the peptidyltransferase reaction and preventing the binding of EF-G. Thiostrepton is specific to Firmicutes because it does not cross the outer membrane of Gram-negative bacterial cells.
Most thiostrepton-resistant mutants are missing the L11 ribosomal protein from the 50S ribosomal subunit. This protein seems not to be required for protein synthesis but plays a role in guanosine tetraphosphate synthesis (see chapter 12). Other mutations confer resistance by changing nucleotides in the 23S rRNA close to where the antibiotic binds. Genes de rived from plasmids and transposons can confer resistance by directing specific methylation of 23S rRNA. Eukaryotes may be insensitive to the antibiotic because the analogous ribose sugars of the eukaryotic 28S rRNAs are normally extensively methylated.
Inhibitors of Binding of aa-tRNA to the A Site
Tetracycline was one of the first antibiotics identified. It may inhibit translation by allowing the aa-tRNA–EF-Tu complex to bind to the A site of the ribosome and allowing the GTP on EF-Tu to be cleaved to GDP, but then inhibiting the next step, causing a futile cycle of binding and release of the aa-tRNA from the A site.
Tetracycline has been a very useful antibiotic for treating bacterial diseases, although it is somewhat toxic to humans because it also inhibits the eukaryotic translation apparatus. Unfortunately, overuse has led to the spread of resistance, and it is no longer useful against many infections. In some types of bacteria, ribosomal mutations confer low levels of resistance to tetracycline by changing protein S10 of the ribosome. However, most clinically important resistance to tetracycline and its derivatives is acquired on plasmids and transposons. One of these genes, tetM, carried by Tn916 and its relatives (see chapter 5), encodes an enzyme that confers resistance by methylating certain bases in the 16S rRNA. Other tetracycline resistance genes, such as the tet genes carried by transposon Tn10 and plasmid pSC101 of E. coli, confer resistance by pumping tetracycline out of the cell. One of the more interesting types of resistance to tetracyclines is due to the so-called ribosome protection proteins, represented by TetO and TetQ, which bind to the A site of the ribosome and release tetracycline from its binding site. This is the type of resistance exhibited by the soil bacteria that make tetracycline.
Inhibitors of Translocation
Aminoglycosides
Kanamycin and its close relatives neomycin and gentamicin are members of a larger group of antibiotics, the aminoglycoside antibiotics, which also includes streptomycin. They seem to affect translocation by binding to the A site of the ribosome. Aminoglycosides have a very broad spectrum of action, and some of them inhibit translation in plant and animal cells, as well as in bacteria. However, their toxicity, especially during sustained use, and high rates of resistance somewhat limit their usefulness as therapeutic agents.
Bacterial mutants resistant to aminoglycosides arise primarily due to genes exchanged on transposons and plasmids. The products of some of these genes inactivate the aminoglycosides by phosphorylating, acetylating, or adenylating (adding adenosine to) them. For example, the neo gene for kanamycin and neomycin resistance, from transposon Tn5, phosphorylates these antibiotics.
Fusidic Acid
Fusidic acid specifically inhibits EF-G (called EF-2 in eukaryotes), probably by preventing its dissociation from the ribosome after GTP cleavage. It has been very useful in studies of the function of ribosomes. In E. coli, mutations that confer resistance to fusidic acid are in the fusA gene, which encodes EF-G. Unexpectedly, some acetyltransferases that confer resistance to chloramphenicol also bind to and inactivate fusidic acid.
A variety of physical techniques, combined with much indirect information accumulated over the years from genetics and biochemistry, have revealed many details of the overall structure of the ribosome. The crystal structures of the individual subunits and the entire 70S ribosome have been determined and correlated with the earlier indirect information. Many laboratories participated in this project, and this awesome achievement will go down in history as one of the major milestones in molecular biology, recognized by the Nobel Prize. We can review only a few of the most salient features here.
Figure 2.21 The composition of a bacterial ribosome containing one copy each of the 16S, 23S, and 5S rRNAs, as well as many proteins. The proteins of the large 50S subunit are designated L1 to L31. The proteins of the small 30S subunit are designated S1 to S21. The 30S and 50S subunits combine to form the 70S ribosome, which carries out protein synthesis.
The two subunits of the ribosome are frequently represented as ovals, with a flat side that binds to the other subunit, leaving a small gap between them. It is through this gap that the mRNA moves, and the aa-tRNAs enter, interact with the mRNA, and pass through the ribosome, contributing their amino acids to the growing polypeptide chain. The newly synthesized polypeptide chain exits through a channel running through the 50S subunit. This channel is long enough to hold a chain of about 70 amino acids, so a polypeptide of this length must be synthesized before the N-terminal end of a protein first emerges from the ribosome.
The rRNAs play many of the most important roles in the ribosome, and the ribosomal proteins seem to be present mostly to give rigidity to the structure, helping to hold the rRNAs in place. This has contributed to speculation that RNAs were the primordial enzymes and that proteins came along later in the earliest stages of life on
Earth. The 23S rRNA, rather than a ribosomal protein, acts as the peptidyltransferase enzyme that performs the enzymatic function that forms the peptide bonds between the carboxyl end of the growing polypeptide and the amino group of the incoming amino acid. Thus, 23S rRNA is an RNA enzyme, or ribozyme. The 23S rRNA also forms most of the channel in the 50S subunit through which the growing polypeptide passes. The 16S RNA plays crucial roles in translation initiation and in matching each incoming aa-tRNA with the mRNA. A structure of the ribosome is shown in Figure 2.22.
Overview of Translation
The information in mRNA is interpreted by the pairing of consecutive triplets of nucleotides (codons) in the mRNA with the complementary anticodon sequences of the corresponding tRNAs. This pairing takes place on the ribosome, and accuracy of translation also depends on matching of a specific tRNA (with the appropriate anticodon) to the corresponding amino acid by a set of enzymes called aminoacyl-tRNA synthetase (aaRSs). aa-tRNAs generated by the aaRS enzymes are delivered to the ribosome by a protein factor called elongation factor-Tu (EF-Tu). During translation, the ribosome moves along the mRNA, and tRNAs interact with the mRNA at three distinct sites, designated the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site (Figure 2.23).