UNIT 08: Antibiotics which inhibit protein synthesis

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The bacterial ribosome is a major target for the action of many clinically useful antibiotics. The selective action of these antibiotics is based mainly on the systematic differences between procaryotic, i.e., bacterial, 70S ribosomes, and eucaryotic, 80S ribosomes. The distinction is not absolute in that eucaryotic cells contain, sequestered within their mitochondria and chloroplasts, ribosomes which resemble, structurally and functionally, the ribosomes which are found in procaryotes.

Other systematic differences between procaryotes and eucaryotes involving non-ribosomal components of the protein synthesizing machinery of the cell also serve as the basis for selective toxicity towards bacterial cells. These include the inhibition of amino acid activating enzyme function as well as auxilliary proteins (initiation and elongation factors) which assist the ribosome in synthesizing proteins.

Targets of antibiotic action in bacterial protein synthesis

Amino acid activation

Amino acid activation enzymes

tRNAs

Peptide bond formation

Ribosome

30S subunit - 800 kD

16S rRNA - 1,500 nt

ribosomal proteins - 20

50S subunit - 1,700 kD

23S rRNA - 3000 nt

ribosomal proteins - 35

Soluble factors

initiation factors - IF1, IF2, IF3

elongation factors - eFTu, eFTs

Mechanisms of resistance

Chloramphenicol - CAT reporter gene system

Tetracycline - Tet selection for resistance, Fusaric acid selection for sensitivity

Streptomycin - dominant/ partially dominant

Erythromycin - dominant/ partially dominant, target modification

Streptogramins A, B - drug modification

Thiostrepton - target modification

Relation to antibiotic production

Conceptual

Procaryotic ribosomes

Eucaryotic ribosomes

Organelle - mitochondrial, chloroplast ribosomes

Example - Erythromycin

1. Erythromycin inhibits bacterial protein synthesis. It belongs to a group of macrocyclic lactone antibiotics called macrolides. There are three major groups of macrolides that inhibit protein synthesis and they contain either 12-, 14-, or 16-membered lactone rings. Erythromycin belongs to the 14-membered ring group. The macrolides, in turn, belong to a superfamily consisting of three chemically distinct groups of antibiotics, the macrolides, lincosamides, and streptogramin B-type (MLS) antibiotics. Erythromycin inhibits protein synthesis by the ribosomes either from bacterial cells or from organelles, such as mitochondria and chloroplasts, of eucaryotic cells. Erythromycin does not inhibit protein synthesis by cytoplasmic ribosomes of eucaryotic cells.

Erythromycin also acts as a motilin agonist and an inducer of liver cytochrome P-450. Motilin is a 22 amino acid peptide whose structure resembles that of erythromycin, at least partially, in three dimensions - peptidomimetic.

2. 70S Ribosomes from bacterial cells contain 2 subunits - 30S and 50S; erythromycin specifically binds to ribosomes with a 1:1 stoichiometry and inhibits a function of the 50S ribosome subunit.

Cytoplasmic ribosomes from eucaryotic cells are generally described as having an 80S sedimentation coefficient, and the subunits, 40S and 60S sedimentation coefficients. Organelle ribosomes are smaller than both. To simplify the discussion, it is often more useful to refer to the "large subunit" and "small subunit" of the ribosome.

3. The 50S ribosome subunit contains 35 proteins and 2 rRNA molecules - the larger rRNA, 23S, is 3000 nucleotides in length and contains nucleotides that are critical for the binding and inhibitory action of erythromycin. The smaller rRNA, 5S, is 120 nucleotides in length.

4. At one time it was thought that the ribosomal proteins performed the reactions involved in protein synthesis and that the rRNA served as an inert scaffold to which the ribosomal proteins were bound in a way that brought them into the required orientation relative to each other. The overwhelming mutational-, footprinting-, and functional data suggest that rRNA plays a more direct role than ribosomal protein in protein synthesis.

5. The 23S rRNA of bacteria can be subdivided into 6 domains based on secondary structure. One of these domains, domain V, 660 nucleotides in length, contains the "peptidyl transferase center" of the ribosome, a 40 nucleotide circle which contains sites critical for peptide bond synthesis.

6. Puromycin contains an amino acid (p-MeO-phenylalanine) attached to an adenosine analog. It resembles the acceptor end of tRNA and is used in model systems to check the effect of other antibiotics on peptide bond formation. Puromycin enters protein synthesis and functions as a minimalist substrate generating peptidyl puromycin. Formation of peptidyl puromycin in such test systems is inhibited by chloramphenicol, but not by erythromycin. Erythromycin does not inhibit the "puromycin reaction".

7. Peptidyl tRNA accumulates during inhibition of bacterial protein synthesis by erythromycin. In studies with defined homopolymeric mRNA in vitro, only short (<5 residues long) peptides are synthesized in reactions that are inhibited by erythromycin. Thus erythromycin does not inhibit peptide bond synthesis, per se, but prevents the growth of peptide chains beyond a length of ca. 5 amino acid residues. One puzzling observation was that polyuridylic acid-directed polyphenylalanine synthesis was not inhibited by erythromycin, whereas polylysine and polyproline synthesis directed by polyadenylic acid and polycytidylic acid, respectively, was inhibited. More on this later.

8. Cells become resistant to erythromycin by one or more of the following biochemical mechanisms (a) modification of erythromycin (phosphorylation, glycosylation, macrolide ring cleavage by esterase), (b) modification of the receptor (post-transcriptional methylation of a single adenine residue in 23S rRNA, A2058, i.e., adenine at coordinate 2058, located within the peptidyl transferase center of 23S rRNA, or mutation in either rRNA, A2058G, or in ribosomal protein L22, (c) elimination of intracellular erythromycin by an efflux ATPase.

9. Post-transcriptional methylation of a single adenine residue in 23S rRNA (A2058), results in co-resistance to the MLS superfamily of antibiotics that bind to the 50S ribosome subunit and inhibit protein synthesis. As a result of the methylation, MLS antibiotics bind to the ribosome with reduced affinity. Individual members of the MLS antibiotic superfamily compete with each other and with chloramphenicol, not a member of the MLS superfamily, for binding to the ribosome, suggesting overlap or interaction between the respective binding sites of these antibiotics. A2058 is specifically methylated by an adenine N6-methyltransferase (methylase).

10. About 30 Erm methylases have been reported in bacteria - ranging from most of the common pathogenic bacteria to the actinomycetes that produce MLS antibiotics and which therefore require the enzyme for protection against the antibiotics which they produce. Some methylases are constitutively expressed, other are inducible by erythromycin. The regulatory mechanism used, in most cases, by the inducible strains is called "translational attenuation".

It useful to distinguish between translational- and transcriptional control of gene activity, also between repression and attenuation. This gives us four possible combinations, each of which should bring to mind a model system in which the mechanism is used. Can you name them?

11. One model of inducible methylase that has been studied intensively, named ermC, comes from Staphylococcus aureus. Unlike regulation of lacZ by transcriptional repression, ermC is regulated by translational attenuation, a mechanism of regulation without repressors. Induction of ermC involves a conformational isomerization of its message from an inactive to an active form. In the inactive form, the ermC message is folded in a way that does not allow initiation of methylase synthesis because the ribosome binding site and initiator methionine codon of the methylase are sequestered by mRNA secondary structure.

12. The ribosome binding site on mRNA for initiation of methylase synthesis, -GGAG-, as well as the initiator methionine codon of ErmC are uncovered as a result of the rearrangement of a set of interacting inverted complementary repeat elements that reassociate as a result of ribosome pausing at a critical location upstream of the ErmC open reading frame while bound to erythromycin (See accompanying figure). The critical location encodes part of a 19 amino acid peptide MGIFSIFVISTVHYQPNKK.

13. The leader peptide provides us with a useful model system in which to study functional aspects of ribosome activity in the presence of erythromycin and to correlate these observations with genetic- and footprinting data. We can study the effects of erythromycin on leader peptide synthesis by measuring expression of methylase, or more precisely, of ß-galactosidase translationally fused to methylase. To do so, we construct an ermC-lacZ translational fusion and measure ß-galactosidase activity as a function of either erythromycin or of other test inducers, and with various amino acid alterations in the leader peptide.

14. If we substitute a stop codon, UAA, for that of Ser-10, AGC, ermC remains inducible. Thus, induction occurs as a result of ribosomes stopping somewhere upstream of Ser-10. Mutational analysis of this region shows that changes at Ile-6, Phe-7, Val-8, and Ile-9 can seriously reduce inducibility. In the presence of erythromycin, ribosomes probably advance on the ermC message as far as Ile-6 to Ile-9 and become stuck. This is confirmed by footprinting experiments.

The antibiotic pseudomonic acid inhibits isoleucyl tRNA synthetase. Using a model ermC-lacZ translational fusion as an assay system, pseudomonic acid induces ß-galactosidase. What does this mean?

15. Mutation A2058G accomplishes the same functional result as the post-transcriptional methylation of 23S rRNA that takes place at the same position. The mutation A2058G is recessive in E. coli because of the presence of seven 23S rRNA gene copies, and dominant in Mycobacterium intracellulare which has only one copy of a gene for 23S rRNA.

Explain the dominance/recessive property of the mutation A2058G

16. The gene msrA from Staphylococcus epidermidis specifies an ATP-dependent efflux that pumps erythromycin out of the cell. This resistance is dominant.

[Graphic figures will be distributed in lecture]

Bacterial signal transduction regulation

Bacterial signal transduction

His-Asp phosphoryl relay systems

How a-helical peptides interact with lipid membranes

How a-helical peptides interact with other a-helical peptides

Enzyme leakage assays

Other modes of b -lactam resistance regulation

b-lactam resistance regulation

AmpC by transcriptional attenuation in S. aureus

AmpC by positive regulation in Citrobacter spp. (Sanders)

Gram positive

Computer tools for studying a-helical peptides

Tools

Protein Predict server

TM Pred server

Hydrophobic cluster analysis (hca) server

PC-TAMMO

		The bacterial ribosome is a major target for the action of many clinically useful antibiotics. The selective action of these antibiotics is based mainly on the systematic differences between procaryotic, i.e., bacterial, 70S ribosomes, and eucaryotic, 80S ribosomes. The distinction is not absolute in that eucaryotic cells contain, sequestered within their mitochondria and chloroplasts, ribosomes which resemble, structurally and functionally, the ribosomes which are found in procaryotes. Other systematic differences between procaryotes and eucaryotes involving non-ribosomal components of the protein synthesizing machinery of the cell also serve as the basis for selective toxicity towards bacterial cells. These include the inhibition of amino acid activating enzyme function as well as auxilliary proteins (initiation and elongation factors) which assist the ribosome in synthesizing proteins. Targets of antibiotic action in bacterial protein synthesis Amino acid activation Amino acid activation enzymes tRNAs Peptide bond formation Ribosome 30S subunit - 800 kD 16S rRNA - 1,500 nt ribosomal proteins - 20 50S subunit - 1,700 kD 23S rRNA - 3000 nt ribosomal proteins - 35 Soluble factors initiation factors - IF1, IF2, IF3 elongation factors - eFTu, eFTs Mechanisms of resistance Chloramphenicol - CAT reporter gene system Tetracycline - Tet selection for resistance, Fusaric acid selection for sensitivity Streptomycin - dominant/ partially dominant Erythromycin - dominant/ partially dominant, target modification Streptogramins A, B - drug modification Thiostrepton - target modification Relation to antibiotic production Conceptual Procaryotic ribosomes Eucaryotic ribosomes Organelle - mitochondrial, chloroplast ribosomes Example - Erythromycin 1. Erythromycin inhibits bacterial protein synthesis. It belongs to a group of macrocyclic lactone antibiotics called macrolides. There are three major groups of macrolides that inhibit protein synthesis and they contain either 12-, 14-, or 16-membered lactone rings. Erythromycin belongs to the 14-membered ring group. The macrolides, in turn, belong to a superfamily consisting of three chemically distinct groups of antibiotics, the macrolides, lincosamides, and streptogramin B-type (MLS) antibiotics. Erythromycin inhibits protein synthesis by the ribosomes either from bacterial cells or from organelles, such as mitochondria and chloroplasts, of eucaryotic cells. Erythromycin does not inhibit protein synthesis by cytoplasmic ribosomes of eucaryotic cells. Erythromycin also acts as a motilin agonist and an inducer of liver cytochrome P-450. Motilin is a 22 amino acid peptide whose structure resembles that of erythromycin, at least partially, in three dimensions - peptidomimetic. 2. 70S Ribosomes from bacterial cells contain 2 subunits - 30S and 50S; erythromycin specifically binds to ribosomes with a 1:1 stoichiometry and inhibits a function of the 50S ribosome subunit. Cytoplasmic ribosomes from eucaryotic cells are generally described as having an 80S sedimentation coefficient, and the subunits, 40S and 60S sedimentation coefficients. Organelle ribosomes are smaller than both. To simplify the discussion, it is often more useful to refer to the "large subunit" and "small subunit" of the ribosome. 3. The 50S ribosome subunit contains 35 proteins and 2 rRNA molecules - the larger rRNA, 23S, is 3000 nucleotides in length and contains nucleotides that are critical for the binding and inhibitory action of erythromycin. The smaller rRNA, 5S, is 120 nucleotides in length. 4. At one time it was thought that the ribosomal proteins performed the reactions involved in protein synthesis and that the rRNA served as an inert scaffold to which the ribosomal proteins were bound in a way that brought them into the required orientation relative to each other. The overwhelming mutational-, footprinting-, and functional data suggest that rRNA plays a more direct role than ribosomal protein in protein synthesis. 5. The 23S rRNA of bacteria can be subdivided into 6 domains based on secondary structure. One of these domains, domain V, 660 nucleotides in length, contains the "peptidyl transferase center" of the ribosome, a 40 nucleotide circle which contains sites critical for peptide bond synthesis. 6. Puromycin contains an amino acid (p-MeO-phenylalanine) attached to an adenosine analog. It resembles the acceptor end of tRNA and is used in model systems to check the effect of other antibiotics on peptide bond formation. Puromycin enters protein synthesis and functions as a minimalist substrate generating peptidyl puromycin. Formation of peptidyl puromycin in such test systems is inhibited by chloramphenicol, but not by erythromycin. Erythromycin does not inhibit the "puromycin reaction". 7. Peptidyl tRNA accumulates during inhibition of bacterial protein synthesis by erythromycin. In studies with defined homopolymeric mRNA in vitro, only short (<5 residues long) peptides are synthesized in reactions that are inhibited by erythromycin. Thus erythromycin does not inhibit peptide bond synthesis, per se, but prevents the growth of peptide chains beyond a length of ca. 5 amino acid residues. One puzzling observation was that polyuridylic acid-directed polyphenylalanine synthesis was not inhibited by erythromycin, whereas polylysine and polyproline synthesis directed by polyadenylic acid and polycytidylic acid, respectively, was inhibited. More on this later. 8. Cells become resistant to erythromycin by one or more of the following biochemical mechanisms (a) modification of erythromycin (phosphorylation, glycosylation, macrolide ring cleavage by esterase), (b) modification of the receptor (post-transcriptional methylation of a single adenine residue in 23S rRNA, A2058, i.e., adenine at coordinate 2058, located within the peptidyl transferase center of 23S rRNA, or mutation in either rRNA, A2058G, or in ribosomal protein L22, (c) elimination of intracellular erythromycin by an efflux ATPase. 9. Post-transcriptional methylation of a single adenine residue in 23S rRNA (A2058), results in co-resistance to the MLS superfamily of antibiotics that bind to the 50S ribosome subunit and inhibit protein synthesis. As a result of the methylation, MLS antibiotics bind to the ribosome with reduced affinity. Individual members of the MLS antibiotic superfamily compete with each other and with chloramphenicol, not a member of the MLS superfamily, for binding to the ribosome, suggesting overlap or interaction between the respective binding sites of these antibiotics. A2058 is specifically methylated by an adenine N6-methyltransferase (methylase). 10. About 30 Erm methylases have been reported in bacteria - ranging from most of the common pathogenic bacteria to the actinomycetes that produce MLS antibiotics and which therefore require the enzyme for protection against the antibiotics which they produce. Some methylases are constitutively expressed, other are inducible by erythromycin. The regulatory mechanism used, in most cases, by the inducible strains is called "translational attenuation". It useful to distinguish between translational- and transcriptional control of gene activity, also between repression and attenuation. This gives us four possible combinations, each of which should bring to mind a model system in which the mechanism is used. Can you name them? 11. One model of inducible methylase that has been studied intensively, named ermC, comes from Staphylococcus aureus. Unlike regulation of lacZ by transcriptional repression, ermC is regulated by translational attenuation, a mechanism of regulation without repressors. Induction of ermC involves a conformational isomerization of its message from an inactive to an active form. In the inactive form, the ermC message is folded in a way that does not allow initiation of methylase synthesis because the ribosome binding site and initiator methionine codon of the methylase are sequestered by mRNA secondary structure. 12. The ribosome binding site on mRNA for initiation of methylase synthesis, -GGAG-, as well as the initiator methionine codon of ErmC are uncovered as a result of the rearrangement of a set of interacting inverted complementary repeat elements that reassociate as a result of ribosome pausing at a critical location upstream of the ErmC open reading frame while bound to erythromycin (See accompanying figure). The critical location encodes part of a 19 amino acid peptide MGIFSIFVISTVHYQPNKK. 13. The leader peptide provides us with a useful model system in which to study functional aspects of ribosome activity in the presence of erythromycin and to correlate these observations with genetic- and footprinting data. We can study the effects of erythromycin on leader peptide synthesis by measuring expression of methylase, or more precisely, of ß-galactosidase translationally fused to methylase. To do so, we construct an ermC-lacZ translational fusion and measure ß-galactosidase activity as a function of either erythromycin or of other test inducers, and with various amino acid alterations in the leader peptide. 14. If we substitute a stop codon, UAA, for that of Ser-10, AGC, ermC remains inducible. Thus, induction occurs as a result of ribosomes stopping somewhere upstream of Ser-10. Mutational analysis of this region shows that changes at Ile-6, Phe-7, Val-8, and Ile-9 can seriously reduce inducibility. In the presence of erythromycin, ribosomes probably advance on the ermC message as far as Ile-6 to Ile-9 and become stuck. This is confirmed by footprinting experiments. The antibiotic pseudomonic acid inhibits isoleucyl tRNA synthetase. Using a model ermC-lacZ translational fusion as an assay system, pseudomonic acid induces ß-galactosidase. What does this mean? 15. Mutation A2058G accomplishes the same functional result as the post-transcriptional methylation of 23S rRNA that takes place at the same position. The mutation A2058G is recessive in E. coli because of the presence of seven 23S rRNA gene copies, and dominant in Mycobacterium intracellulare which has only one copy of a gene for 23S rRNA. Explain the dominance/recessive property of the mutation A2058G 16. The gene msrA from Staphylococcus epidermidis specifies an ATP-dependent efflux that pumps erythromycin out of the cell. This resistance is dominant. [Graphic figures will be distributed in lecture]

Bacterial signal transduction regulation		Bacterial signal transduction His-Asp phosphoryl relay systems How a-helical peptides interact with lipid membranes How a-helical peptides interact with other a-helical peptides Enzyme leakage assays
Other modes of b -lactam resistance regulation		b-lactam resistance regulation AmpC by transcriptional attenuation in S. aureus AmpC by positive regulation in Citrobacter spp. (Sanders) Gram positive
Computer tools for studying a-helical peptides		Tools Protein Predict server TM Pred server Hydrophobic cluster analysis (hca) server PC-TAMMO