Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance

Home , Carbomycin, Macrolide

Current Topics in Medicinal Chemistry 2003, 3, 949-961 949 Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance

Marne Gaynor and Alexander S. Mankin*

Center for Pharmaceutical Biotechnology – M/C 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607, USA

Abstract: Macrolides are among the most clinically important antibiotics. However, many aspects of macrolide action and resistance remain obscure. In this review we summarize the current knowledge, as well as unsolved questions, regarding the principles of macrolide binding to the large ribosomal subunit and the mechanism of drug action. Two mechanisms of macrolide resistance, inducible expression of Erm methyltransferase and peptide-mediated resistance, appear to depend on specific interactions between the ribosome-bound macrolide molecule and the nascent peptide. The similarity between these mechanisms and their relation to the general mode of macrolide action is discussed and the discrepancies between currently available data are highlighted.

Key words: macrolides, erythromycin, ketolides, azithromycin, clarithromycin., tylosin, carbomycin, spiramycin, ribosome, resistance.

INTRODUCTION transferase center and cause cell growth arrest due to inhibition of protein synthesis [1,2]. However, in spite of Macrolides belong to one of the most commonly used more than 50 years of research, the mode of macrolide families of clinically important antibiotics used to treat inhibition of ribosome activity is understood only in the infections caused by Gram-positive bacteria such as most general terms. Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes. Chemically, macrolides are The extensive use of these antibiotics has led inevitably represented by a 14-, 15- or 16-membered lactone ring to the spread of resistant strains. Expression of some of the carrying one or more sugar moieties and additional resistance determinants is inducible by macrolides. Of substitutions linked to various atoms of the lactone ring, particular interest are Erm methyltransferases, which Fig (1). specifically methylate a unique nucleotide within the macrolide binding site. The mechanism of Erm induction Erythromycin A, a 14-membered ring drug, was the first depends on ribosome stall within the translated regulatory clinically used macrolide. Drug delivery problems resulting open reading frame preceding the Erm cistron, and is from acid instability, prompted the design of newer apparently closely related to the general mode of macrolide macrolides. Increased acid stability as well as an increase in action on protein synthesis. However, the details of the the range of antimicrobial activity characterized the second mechanism of Erm induction are not known. generation, which includes 14-membered ring drugs, such as clarithromycin and roxithromycin as well as the 15- In addition, a novel mechanism of macrolide resistance membered ring azithromycin. In addition, the 16-membered has recently been described which is mediated by the ring macrolides such as tylosin, carbomycin A and expression of specific short peptides in the cell. Although it spiramycin and others also exhibited significant is not entirely clear how expression of short peptides can antimicrobial activity, and were originally thought to be the render cells resistant to macrolides, the underlying principles answer to the growing occurrence of erythromycin resistant maybe related to the mechanisms of inducible resistance and infections. Unfortunately, all of these drugs became prone to the mode of drug-induced inhibition of translation. the selection of resistant strains. The newest generation of macrolides, the ketolides, whose clinical use is in its early In this brief review we try to summarize the current stage, are characterized by improved activity against some of understanding of the mechanism of macrolide action, and the the resistant strains. molecular mechanisms of inducible erm expression and peptide-mediated macrolide resistance. Several reviews It has been well documented that macrolides bind to the dealing with these subjects appeared in the past and we refer large ribosomal subunit in the vicinity of the peptidyl the reader interested in specific details to those publications [1-4]. Instead, we will try to focus on the apparent *Address correspondence to this author at the Center for Pharmaceutical discrepancies and less clearly defined details in the existing Biotechnology – M/C 870, University of Illinois, 900 S. Ashland Ave., models. We strongly believe that understanding these Chicago, IL 60607, USA; Tel: 312-413-1406; Fax: 312-413-9303; E-mail: [email protected] discrepancies exist and finding ways to resolve them will

Erythromycin A Clarithromycin Azithromycin

O O N 9 10 8 OH OH HO 11 HO OMe HO 7 12 OH 6 N OH N OH N 13 5 HO HO HO 4 O O O O 14 O O O O O 1 3 2 OMe OMe O O O O OMe O O

OH OH OH O O O

Telithromycin (HMR-3647) Cethromycin (ABT-773)

N N O N H O O N N O O O N N O O N HO O O O HO O O O O O

O O

O Carbomycin A O Tyl osin N O O O OH O HO O O O O O O OAc O OMe OMe N O HO O OH HO O O O O O OH O OH O

Fig. (1). Chemical structures of the macrolide antibiotics. First row: drugs of the first generation (erythromycin) and second generation (14-membered ring clarithromycin and 15-membered ring azithromycin). Second row: ketolides, the macrolides of the third generation. Third row: examples of 16-membered ring macrolides. help to direct further research focused on the molecular The X-ray structures confirm conclusions drawn from mechanisms of macrolide action and drug resistance. biochemical data, which, indicated that RNA constitutes the primary component of the macrolide binding site, Fig. (2A). A number of nucleotide residues in domain V of 23S rRNA THE MACROLIDE BINDING SITE interact with the macrolide molecule. Important contacts, which contribute markedly to the strength of interaction of The general location of the macrolide binding site on the the macrolide molecule with the ribosome, are formed large ribosomal subunit has been initially mapped using a between the C5 mono- or disaccharide side chains of 14- 15- combination of biochemical and genetic methods [2,5-9]. and 16-membered ring macrolides and rRNA. The Nonetheless, the details of the molecular interactions of the desosamine sugar of erythromycin and other related 14- various classes of macrolides with the ribosome have just membered ring macrolides form hydrogen bond interactions started to emerge with the release of several crystallographic with the nitrogen bases of the nucleotide residues A2058, structures of the archaeal and bacterial large ribosomal A2059 (here and throughout the entire paper we will use E. subunits and their complexes with antibiotics [10-13]. coli nucleotide numbering to facilitate the discussion). The Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 3 base pair 2611-2057, more specifically, the nucleotide Biochemical and genetic data revealed interaction of occupying position 2057, may also be involved in hydrogen various macrolides, including erythromycin, ketolides and bonding with the C5 desosamine of 14-membered ring tylosin, with the loop of helix 35 in domain II of 23S rRNA macrolides (as seen in the bacterial, Deinococcus [7,9]. Mutations or changes in posttranscriptional radiodurans structures) [12], or it may establish modification in this rRNA region confer weak resistance to hydrophobic interactions with the lactone ring (as seen in the macrolides [7,18]. In addition, a bound macrolide either complexes of 15- and 16-membered ring macrolides with the enhanced or decreased accessibility of nucleotides in the archaeal 50S ribosomal subunit) [13]. In addition, the helix 35 loop to chemical modification in the RNA probing desosamine sugar can potentially interact with the backbone experiments [5,7,9,14]. Protection or enhancement of phosphate oxygen of G2505. The mycaminose O2 of the accessibility of A752 to dimethyl-sulfate modification mycaminose-mycarose disaccharide of 16-membered ring appeared to correlate with the chemical structure of the alkyl- macrolides also forms a hydrogen bond with adenine at the aryl side chain of ketolides, suggesting that the side chain position 2058 revealing this nucleotide as one of the main might contact the loop of helix 35 [16], (Xiong and Mankin, binding determinants of all macrolides. The C5 unpublished). Although crystallographic structures show that mycaminose-mycarose disaccharides of tylosin, carbomycin the mucinose side chain of 16-member ring tylosin can A and spiramycin form a number of additional interactions, apparently reach the loop of helix 35 [14], no interactions of mainly of a hydrophobic nature. The interactions of the C5 14-membered ring macrolides of the erythromycin type, sugar residues with positions 2057-2059 explain why ABT-773 or azithromycin with this rRNA region were mutations at these nucleotides or dimethylation of A2058 revealed [12], (Schlünzen and Yonath, unpublished). The can render macrolide resistance [12]. quinolylallyl side chain of ABT-773 forms hydrogen bonds with C1782 in domain IV of 23S rRNA and U790 in Interaction of the lactone ring with the ribosome may domain II. However, the latter interaction it is not close account for more then 25% of the free binding energy of the enough to the loop of helix 35 to explain the protection of drug [14]. Hydrophobic interactions appear to contribute A752 by ABT-773 observed in the RNA footprinting significantly to this interaction. In particular, the lactone experiments [14]. Thus, it remains unclear whether the ring interacts hydrophobically with a crevice formed by ketolide side chain and/or the loop of helix 35 in the crystal rRNA bases 2057-2059 [14]. The crystallographic structures structure are positioned exactly as in the “live and breathing” of complexes of the 16-membered ring macrolides, tylosin, ribosome. Altogether, crystallographic studies did not show carbomycin A and spiramycin, with the large ribosomal any strong interactions of the ketolide side chain with the subunit of Haloarcula marismortui [13] suggest that the ribosome, leaving the 11, 12-carbamate and its interaction acetaldehyde group at C6 position of the lactone ring forms with U2609 as potentially the major factor enhancing a reversible covalent bond with the N6 of A2062, which ketolide binding. may contribute to the binding energy of these drugs. This interaction is not possible for the 14-membered ring A macrolide molecule is coordinated in its binding site macrolides of the erythromycin group or the 15-member ring by multiple hydrophobic and hydrogen bonds (and possibly, azithromycin, which carry either a hydroxyl or an ester at the a covalent bond in case of some 16-membered ring C6 position of the lactone ring. This explains why 2062 macrolides) between its functional groups and 23S rRNA. mutation confers resistance specifically to 16-member ring, These interactions with RNA account for most of the free but not 14- or 15-membered ring macrolides [15]. energy of drug binding. In addition, some macrolides can reach ribosomal proteins L4 and/or L22. The mucinose sugar Ketolides are the most recently introduced generation of of tylosin interacts with L22 and the furosamine residue of macrolides. They are characterized by the replacement of the spiramycin interacts with L4 [14]. The proximity of these C3 cladinose sugar with a keto group. In addition, most proteins to the macrolide binding site explains why clinically relevant ketolides also contain extended alkyl-aryl mutations in L22 and L4 protein genes can render cells side chain appendages as well as an 11, 12-carbamate cycle. resistant to macrolides [19-22]. Ketolides exhibit increased binding to the ribosome compared to macrolides of previous generations [16,17]. In When analyzing crystallographic structures of the contrast to expectations, the recently solved structure of a ribosome-macrolide complexes, it is important to keep in complex of a ketolide, ABT-773, with the D. radiodurans mind that although the general features of the drug-ribosome ribosome (Schlünzen and Yonath, personal communication), interactions observed with the 50S subunit of a halophilic did not provide clear clues into the molecular determinants archaeon (H. marismortui) and a bacterium (D. radiodurans) of increased ketolide affinity. The 11-OH and 12-OH lactone are in general agreement, some details vary. For example, hydroxyls of erythromycin and related drugs could form the precise position and conformation of the lactone ring of hydrogen bonds with O4 of U2609. The carbamate cycle that several macrolides seen in structures solved by the Yonath replaces these hydroxyls in ABT-773 (and other ketolides) is group [12] differ from those seen by the Steitz laboratory seen interacting with O4 of U2609 in D. radiodurans 50S [14]. At this point it is unclear whether the discrepancies are subunits. Biochemical data show that this interaction is due to uncertainty in the crystallographic structures, more important for ketolides than for the drugs of the differences in the ribosomes used, or the difference in erythromycin group because the mutation of U2609 to C crystallization procedures (co-crystallization versus crystal confers resistance specifically to ketolides but not other soaking). It is important to note, that in the 23S rRNA of macrolides [14]. However, crystal structures did not provide H. marismortui several positions involved in macrolide a clear explanation of the significance of this mutation. binding are different from those in the bacterial 23S rRNA. 4 Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 Gaynor and Mankin

These include such important positions as 2058 (A in The main mechanism of inhibition of protein synthesis bacteria, G in archaea) and 2609 (U in bacteria, C in H. by macrolides is related to their binding in the nascent marismortui). The nucleotide sequence differences might peptide exit tunnel. The exit tunnel is formed primarily by potentially affect the affinity of the macrolide molecule to 23S rRNA. It starts at the peptidyl transferase center and the ribosome and precise molecular interactions in the spans the entire body of the subunit finally opening at its binding site. Indeed, the mutation of G2058 to A in the “back” [10,30-32]. Although originally viewed as an inert closely related archaeon, Halobacterium halobium, signifi- conduit for nascent peptides of any sequence synthesized by cantly increases sensitivity of the mutant to erythromycin the ribosome, the tunnel is now considered an active and and other macrolides (Mankin, Xiong, Tait-Kamradt, dynamic functional entity [33]. Several studies showed that unpublished). However, even the drug complexes with the interactions between the ribosome and the nascent peptide bacterial ribosome of D. radiodurans should be considered that take place inside the exit tunnel affect the progression of cautiously: having only one bacterial structure is not protein synthesis as well as the reactions catalyzed by the sufficient to distinguish between the general and species- ribosomal peptidyl transferase [34,35], (see [36] for review). specific determinants of macrolide binding. For example, The tunnel is relatively wide (15 Å average). However, it two molecules of azithromycin were found bound to the D. contains a constriction, (ca. 10 Å wide) formed by proteins radiodurans ribosome in the co-crystallization experiments. L4 and L22, which is located a short distance from the One of the azithromycin molecules occupied the “conven- peptidyl transferase center. Macrolides bind close to this tional” site while the other is seen bound immediately next constriction and pose a molecular road block for the growing to it, making direct contact with the proteins L4 and L22 as polypeptide chain [12,13,33]. In the presence of bound 14- well as domain II of 23S rRNA. The significance of this and 15-membered ring macrolides, polymerization of the second site with regard to azithromycin inhibitory action first several amino acids continues unperturbed and remains unclear. It could possibly be a species-specific inhibition of polypeptide growth occurs only after the feature because the amino acid Gly60 in protein L4 that is nascent peptide becomes large enough to reach the bound contacted by the drug is unique in D. radiodurans compared macrolide near the tunnel constriction. The inhibition of to other bacteria (Franceschi, personal communication). peptide progression eventually results in the dissociation of peptidyl tRNA from the ribosome. The latter apparently takes place during an attempted act of translocation during LOCATION OF THE MACROLIDE BINDING SITE which the contacts of tRNA with the ribosome must be IN THE RIBOSOME AND THE MECHANISM OF “loosened”. ACTION This mechanistic model explains biochemical The precise mechanism of protein synthesis inhibition by observations regarding macrolide activity and is generally macrolides depends on the specific chemical structure of the consistent with the macrolide location in the ribosome drug molecule. This affects its interaction with the ribosome [24,25,37]. However, the main question that remains as well as the mode of the inhibitory action. Four modes of unresolved is the length of the nascent peptide that the inhibition of protein synthesis have been ascribed to macrolide-bound ribosome can synthesize. In poly (A)- macrolides: 1) Inhibition of the progression of the nascent dependent cell-free translation systems, macrolides were peptide chain during early rounds of translation [23,24]; 2) reported to cause accumulation of di-lysine but inhibited Promotion of peptidyl tRNA dissociation from the ribosome synthesis of longer peptides. In poly (U)-dependent protein [25]; 3) Inhibition of peptide bond formation [23]; and 4) synthesis, accumulation of Phe2 and Phe3 was observed in Interference with 50S subunit assembly [26]. All of these the presence of erythromycin, while formation of longer mechanisms have some correlation with the location of the peptides was inhibited [28,29]. Also noteworthy is the macrolide binding site on the ribosome. observation that the exact point of erythromycin inhibition of elongation appears to be dependent on the amino acid The macrolide binding site is located on the large sequence of the nascent peptide. While the drug does not ribosomal subunit inside the nascent peptide exit tunnel near affect transfer of Ac-Gly-Gly to puromycin, the transfer of the peptidyl transferase center, Fig. (2B and 2C). Its Ac-Pro-Gly is efficiently inhibited [23]. The effect of proximity to the peptidyl transferase center explains the erythromycin on elongation of very short peptides appears to inhibitory effect of some macrolides on peptide bond contradict more recent observations using an in vitro formation. The sugar residues attached at the C5 position of translation system driven by synthetic mRNAs, in which the lactone ring protrude towards the peptidyl transferase erythromycin caused the accumulation of peptidyl-tRNAs center. The long disaccharide mycaminose-mycarose side carrying significantly longer - 6 to 8 amino acid residue-long chains of the 16-membered ring drugs tylosin, spiramycin peptides (Tenson, personal communication). Furthermore, and carbomycin A stretch far enough towards the active site the ketolide telithromycin allowed the ribosome to of the peptidyl transferase to directly interfere with the synthesize nascent peptides that were even longer – 9 to 12 catalysis of peptide bond formation [13,27]. The shorter amino acids long. Modeling studies suggest that 6 to 8 desosamine monosaccharide residues of the 14-membered amino acids can potentially fit between the peptidyl ring macrolides do not reach the peptidyl transferase, which transferase active site and the macrolide roadblock [12]. If explains the lack of inhibitory effects of these drugs on the so, then how can erythromycin affect elongation of a di- reaction of transpeptidation [24,28,29]. In contrast, some peptide? And how can a 12-amino-acid-long nascent peptide mild stimulation of peptidyl transfer by erythromycin is synthesized in the presence of ketolides fit? Is it folded in a observed in the puromycin reaction [23]. specific way within the ribosome? Another question that Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 5

Fig. (2). Molecular interactions and location of the macrolide binding site in the large ribosomal subunit. A. Nucleotide environment of a macrolide (erythromycin) in the 50S ribosomal subunit of the bacterial (Deinococcus radiodurans) ribosome [12]. The erythromycin molecule is shown red. Nucleotide residues are numbered as in E. coli 23S rRNA. B. Position of erythromycin in the nascent peptide exit tunnel, front view. 23S rRNA is shown light purple, 5S rRNA is green, ribosomal proteins are dark purple and erythromycin red. The view is from the interface of the large ribosomal subunit down the peptidyl transferase center. C. Position of erythromycin in the nascent peptide exit tunnel, side view. rRNA and ribosomal proteins are shown light gray and dark gray, respectively. The P-site-bound tRNA is shown blue (modeled from [61]), modeled nascent peptide is gold and erythromycin molecule is red. View is from the side of the L11 stalk. (Fig. (2) was prepared by Dr. Joerg Harms (Max-Planck Research Unit for Ribosomal Structure) using RIBBONS [62]). 6 Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 Gaynor and Mankin remains unclear is whether the length of the nascent peptide binding to the ribosome [21,22]. Mutations in another produced in the presence of erythromycin or related ribosomal protein, L22, do not significantly perturb drug macrolides depends on the peptide amino acid composition affinity, but appear to act through an indirect mechanism. and/or sequence. One would expect that the growth of a These mutants exhibit a wider opening of the tunnel so that nascent peptide composed of bulky amino acids would be the nascent peptide can apparently “slip by” the macrolide arrested earlier than that of a peptide composed of smaller molecule bound in the tunnel (and/or possibly displace the residues. Furthermore, since the macrolide molecule and the drug) [21,33]. Mutations in ribosomal protein genes are a nascent peptide appear to be competing for the same space in frequent cause of drug resistance because a single mutational the exit tunnel, it is possible that certain peptides might event is sufficient to render cells resistant to a macrolide. In exhibit affinity for the components of the tunnel wall that contrast, resistance due to rRNA mutations is less common interact with a macrolide molecule, and could competitively [44]. Because of the multiplicity of rRNA genes in the remove macrolide from its binding site. In this scenario, majority of bacteria, the beneficiary (for the pathogen) effect synthesis of different proteins can be inhibited to a different of the mutation in one rRNA gene copy is usually masked extent by macrolides and such specificity may depend both by the abundance of wild type rRNA transcribed from on the structure of the drug and the structure of the nascent unmutated gene copies. Therefore, this mechanism of peptide and their interactions with the ribosome. resistance is more often found in the organisms with a low copy number of rRNA cistrons (for example, Helicobater Interference with ribosome assembly may contribute pylori that has two rRNA operons) (see [2,3] for review). further to the overall inhibitory activity of macrolides [38,39]. In the presence of macrolides, precursors of 50S The most frequently found mechanism of macrolide ribosomal subunits accumulate. These precursors do not binding site modification is dimethylation of a single 23S contain a full protein complement of the 50S subunit and rRNA nucleotide, A2058, by Erm-type methyltransferases. sediment in a sucrose gradient as 30S particles [40]. The The erm genes are found in macrolide producers from which 23S rRNA in 50S precursor particles is more accessible for they were apparently disseminated to the clinical pathogens chemical modification than in the fully assembled 50S through horizontal gene transfer [45]. Dimethylation of subunits (Xiong, Champney and Mankin, unpublished). A2058, which is located within the macrolide binding site, Although the location of the macrolide binding site in the drastically decreases drug affinity due to steric hindrance assembly intermediates remains to be determined; it is thus, rendering bacteria resistant to high concentrations of conceivable that binding of the macrolide to the subunit macrolide antibiotics [12,16,46]. A2058 is also intimately precursor (in its “conventional” site or a different site) may involved in the binding of lincosamides and streptogramin prevent specific conformational rearrangements and/or B [12]. Hence, Erm-based methylation (termed MLSB protein binding required for the completion of assembly resistance) renders the cell resistant to at least three unrelated [41]. A correlation between the inhibition of protein classes of antibiotics. Importantly, the fully assembled synthesis and inhibition of ribosome assembly suggests that ribosome is not a substrate for erm methylation because not only drug binding to the precursor, but also either A2058 is buried deep inside the large ribosomal subunit and general reduction of protein synthesis or inhibition of apparently is not accessible to Erm methyltransferase [47]. production of a specific protein(s) (for example, chaperons Methylation of A2058 can take place only during ribosome which may contribute to ribosome biogenesis [42,43]) is assembly, which leaves a very narrow time window for the required for inhibiting assembly of the 50S subunit by erm enzyme to methylate its rRNA target. macrolides. The importance of assembly inhibition for the overall inhibitory action of macrolides remains to be Expression of erm genes can either be constitutive or determined. Since inhibition of the assembly takes place inducible. Inducible erm-based resistance, where production within the background of the arrested protein synthesis, it is of Erm methyltransferase is activated only when cells are unclear whether reduced production of new ribosomes exposed to the drug, is found in some antibiotic producers amplifies the cell’s “suffering” when protein synthesis is as well as in many clinical pathogens. The frequent already being inhibited. occurrence of inducibly expressed erm [48,49] suggests that A2058 dimethylation is apparently “unhealthy” for unperturbed protein synthesis. Bacteria appear to avoid MACROLIDE RESISTANCE DUE TO THE TARGET unnecessary methylation of this nucleotide unless it becomes SITE MODIFICATION essential for survival in the presence of the drug.

High efficacy and safety of macrolides as well as their The molecular mechanisms of Erm induction are related use as an alternate therapy for penicillin-intolerant patients to the general mode of macrolide action, but they are largely made them popular drugs. The wide use of these antibiotics enigmatic. The inducible erm cassette consists of two led inevitably to the spread of the resistance strains. The two functional parts – the Erm gene whose expression is most common mechanisms of resistance are excretion of the normally repressed due to sequestration the erm ribosome drug from the cell and modification of the drug target site binding site in the mRNA secondary structure and a [2]. The latter mechanism comes in the form of a site- constitutively translated leader peptide cistron that precedes specific posttranscriptional modification of 23S rRNA or erm [50,51], Fig. (3A). In the presence of erythromycin, the mutations in 23S rRNA or ribosomal proteins. ribosome is believed to stall around the 8th –9th codon of the leader peptide open reading frame (ORF). The stall of the Mutations in protein L4 directly or allosterically affect ribosome triggers a conformational rearrangement in mRNA macrolide binding and cause resistance by preventing drug resulting in the opening of the erm ribosome binding site Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 7 and thus, activation of erm translation, Fig. (3B) [52,53]. resistance may be delayed. Moreover, only newly assembled Since stall on the leader ORF occurs only in the presence of ribosomes can carry methylated 23S rRNA, as assembled the drug, the ribosome translating the leader peptide must ribosomes are not erm methlyase targets (see above). Thus, bind a macrolide molecule. On the other hand, the ribosome the ability of a macrolide to induce erm resistance may that will translate the erm cistron, should be drug free at depend on the kinetic interplay of drug binding and least during early steps of erm translation in order to be able dissociation from the ribosome and the rate of translation. It to synthesize the entire polypeptide. This implies that is conceivable that macrolides with a high off-rate and a efficiency of erm induction should depend critically on moderate-affinity binding will induce erm expression at a macrolide concentration. At very low drug concentrations, broader range of drug concentrations, while the tightly too few ribosomes will carry the antibiotic and thus stalling binding macrolides with a slow off-rate will be less-efficient on the leader ORF will not occur. On the other hand, if the inducers. Indeed, the ketolides, which bind much tighter to drug concentration abruptly becomes very high, translation the ribosome than macrolides appear to be poor inducers of of the Erm cistron maybe inhibited and expression of the erm [16,17,54].

Lys A Lys

Asn

Pro S.D.E Gln 1234Tyr His Met Val S.D.L Met Gly Ile Phe Ser Ile Phe Val Ile Ser Thr Erm

2 3

S.D. Met S.D.L Met Gly Ile Phe Ser Ile Phe Val Ile Ser Thr Val His Tyr GLn Pro Asn Lys Lys E Erm

Fig. (3). Inducible Erm resistance. The secondary structure of the leader region of the bi-cistronic mRNA coding for the leader peptide and Erm methyltransferase in the non-induced (A) and induced (B) configuration [51]. The sequence of the short peptide encoded in the leader open reading frame and its Shine-Dalgarno region (S.D.L) are shown. Shine-Dalgrano region (S.D.E) of the Erm cistron and the location of Met initiator codon are indicated. In the presence of an inducing macrolide antibiotic, the ribosome stalls around 8th-9th codon of the leader cistron. Stalling causes rearrangement of the mRNA secondary structure which renders the translation initiation region of the Erm cistron accessible. 8 Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 Gaynor and Mankin

The critical and the least understood component of the is reminiscent of interactions that were proposed to take mechanism of erm induction is the dependence of ribosome place in a completely different mechanism of macrolide stall on both the sequence of the leader nascent peptide and resistance. In this mechanism, resistance to macrolides is the chemical nature of the inducing macrolide. This suggests mediated by expression of specific peptides, 4-6 amino acid- that a specific interaction between the peptide and the bound long [56,57] (see [4] for review). Biochemical evidence macrolide molecule is required for ribosome stall [51]. Since indicates that the peptides act in cis, rendering the ribosome the latest experiments show that macrolides (erythromycin) on which they were translated refractory to inhibition by normally inhibit ribosome progression when the nascent macrolides. Screening of random 5-codon-long mini-gene peptide is between 2 and 8 amino acids long (Tenson, libraries showed that only mini-genes coding for peptides personal communication), it appears that the ribosome with a specific amino acid sequence could confer resistance, should stall at approximately the correct place in the leader Fig. (4). Thus, peptides conferring the highest resistance to peptide ORF to elicit induction, irrespective of the leader erythromycin (E-peptides) are characterized by the consensus peptide sequence. However, this is not true. The identity of sequence M-(L)-L/I-(F)-V where the third position is almost leader peptide amino acids 5-9 (S-I-F-V-I) is critical for always occupied by Leu or Ile, while the second and fourth induction [55]. Since these C-terminus-proximal residues, positions show strong preference for hydrophobic amino but not the N-terminus of the nascent peptide appear to acids, more frequently Leu/Ile and Phe, respectively [57]. determine the efficiency of ribosome stall, the nascent Although cells expressing E-peptides also exhibit some peptide in the stalled complex might be folded in a specific resistance to ketolides, the peptides conferring the highest way to allow direct contact between the critical amino acids ketolide resistance (K-peptides) conform to a different and the drug Fig. (5A). The immediate functional conse- consensus: M-K/R-(F/L/V)-X-X, where X indicate positions quences of such interactions are not clear. One possibility is which do not show an obvious amino acid preference [58]. that stall occurs because dissociation of peptidyl-tRNA is Peptides with yet different consensus sequences render cells inhibited due to a specific drug-nascent peptide interaction. resistant to the 14-membered ring drugs oleandomycin and However, other mechanisms are possible. troleandomycin, 15-membered ring azithromycin, 16- membered ring josamycin and other macrolides [4] and (Gaynor, Mankin and Tenson, unpublished). The strong PEPTIDE-MEDIATED MACROLIDE RESISTANCE. correlation between the sequence of the resistance peptide and the structure of the drug suggests that a direct interaction The potential structure-specific interaction between the between the drug and the peptide takes place on the nascent peptide and the macrolide on the ribosome, which ribosome. The current model proposes that the newly may be the key component of the erm induction mechanism, synthesized resistance peptide interacts with the ribosome-

E-peptides K-peptides

ATG CTT TTA CGT ATC TAA M L L R I ATG AAA TTA AAA CTC TAA MM KK LL KK LL ATG ACA TTA AAA GTC TAA M T L K V ATG AAA CTG AAG CTC TAA MM KK LL KK LL ATG ATT CTA AAG TTA TAA M I L K L ATG AAA ATG AAA GTT TAA MM KK MM KK VV ATG ATG CTA AAA TTG TAA M M L K L ATG AAA ATG AAA CTC TAA MM KK MM KK LL ATG CTG CTT ACG GTA TAA M L L T V ATG CGC TTT TTT GTC TAA MM RR FF FF VV ATG CTG TTG TTG GTG TAA M L L L V ATG CGG TTC TTT GTT TAA MM RR FF FF VV ATG CTG TTG CTG GTG TAA M L L L V ATG CGG TTC TTT GCT TAA MM RR FF FF AA ATG CTG CTA TTG GTT TAA M L L L V ATG AAG TTC TTT GTT TAA MM KK FF FF VV ATG GTT TTG TTT GTT TAA M V L F V ATG CGA GTA TAC CGA TAA MM RR VV YY RR ATG CGT TTA TTT GTT TAA M R L F V ATG AGG CGT TTT ATT TAA MM RR RR FF II ATG TTA CTT TGG GTT TAA M L L W V ATG CTT CGT TGG TGG TAA MM LL RR WW WW ATG GTA ATT TTG GTA TAA M V I L V ATG GTA ATT TTG GTA TAA M V I L V ATG TTA ATC ACA GTA TAA M L I T V ATG GCT TTA AAA TAC TAA M A L Y T ATG GTA CAA ACA GTA TAA M V Q T V ATG GTA TAC ACA ATC TAA M V Y T I

Fig. (4). Short peptides conferring resistance to erythromycin (E-peptides) or ketolide ABT-773 (K-peptides) selected from a random five-codon-long mini-gene library [57]. The nucleotide sequences of the mini-genes and the amino acid sequences of the encoded peptides are shown. Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 9 bound macrolide molecule and evicts the drug molecule FUTURE DIRECTIONS from its binding site on the ribosome therefore allowing the ribosome to get re-engaged, at least temporarily, in We mentioned some main achievements and problems in translation of cellular proteins [58]. Since the size of the understanding the mechanism of action of macrolide peptide appears to be important [56], the act of translation antibiotics and mechanisms of macrolide resistance. termination (release of the nascent peptide from peptidyl- However, it is clear that comprehensive knowledge of the tRNA) and/or subsequent events may contribute to the drug principles of drug interaction with the ribosome and the displacement [59]. molecular mechanisms of drug resistance are essential prerequisites for the development of better drugs. The atomic The parallel between the mechanism of erm induction structures of the ribosome and the ribosome-drug complexes and peptide-mediated macrolide resistance is obvious. In will definitely aid the rational design of new macrolide both cases, the mode of ribosome function appears to depend derivatives, which might exhibit tighter binding to the directly on interaction between the short nascent peptide and target. It is unclear, however, whether it is possible to the drug molecule bound in the exit tunnel. Strikingly, the design a molecular platform that would strengthen the consensus sequence of the pentapeptides that render cells interaction of the lactone ring and its side chains with the resistant to erythromycin, M-(L)-L/I-(F)-V, has a clear ribosome while avoiding steric clash with the A2058 resemblance to the C-terminal amino acids of the erm leader dimethyl moiety of an erm-methylated ribosome. peptide in the stalled complex, (… I-F-V). It is tempting to think that this is not a mere fortuitous coincidence and that The mechanism of macrolide action is only starting to the two mechanisms exploit similar interactions between the emerge. Again, the precise knowledge of the mode of drug and the nascent peptide on the ribosome, Fig. (5). macrolide binding to the ribosome will be instrumental in However, the consequences of such interactions may be understanding how various macrolides affect different aspects different – ribosome stalling during erm induction and drug of protein synthesis. The length of the nascent peptide dissociation (probably accompanied by the release of the synthesized in the presence of macrolides, and the relation ribosome from the mini-gene transcript) in the case of E- between inhibition of elongation and the nascent peptide peptide expression. How either of these events may occur sequence need to be investigated. Kinetics and mechanisms remains unclear. of peptidyl-tRNA dissociation induced by macrolides also require more detailed biochemical experimentation. The AB

Met Gly Release factor Ile Val Ile Val Phe Phe Phe Ser Ile Leu Val Met

Fig. (5). Putative similarity of the mechanisms of Erm induction and peptide-mediated macrolide resistance. A. Schematic representation of the ribosome stalled on the Erm leader cistron. The ribosome is shown with the 8th (Val codon) in the P-site and the 9th (Ile) codon in the A-site. Erythromycin (black octagonal) is shown bound near the constriction of the exit tunnel. The amino acid residues whose identities are important for ribosome stall are shown gray. The model reflects our proposal that the nascent peptide may fold back in order to allow direct contact between the critical amino acid residues and the macrolide. B. The pre-termination complex formed by the ribosome which has finished translation of a resistance peptide. The sequence of one of the E-peptides (see Fig. (4)) is shown. According to the “bottle-brush” model [58], the release of the E-peptide will promote dissociation of the bound macrolide (in this case, erythromycin) from the ribosome. The amino acid residues whose identities are important to confer erythromycin resistance are shown in gray. 10 Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 Gaynor and Mankin release of the ribosome from mRNA, resulting from [3] Vester, B.; Douthwaite, S. Macrolide resistance peptidyl-tRNA dissociation, should potentially allow this conferrred by base substitutions in 23S ribosomal RNA. ribosome to re-engage in protein synthesis. One would Antimicrob. Agents Chemother. 2001, 45, 1-12. wonder, therefore, if blocking the exit tunnel farther away [4] Tenson, T.; Mankin, A.S. Short peptides conferring from the peptidyl transferase center would produce a more resistance to macrolide antibiotics. Peptides 2002, 22, efficient inhibition of protein synthesis. The longer nascent 1661-1668. peptide stuck in the exit tunnel may prevent peptidyl-tRNA from dissociating and thus, could irreversibly incapacitate [5] Moazed, D.; Noller, H.F. Chloramphenicol, erythromycin, the large ribosomal subunit. carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA. Though the general inhibition of protein synthesis by Biochimie 1987, 69, 879-884. macrolides is obviously an important factor in cell growth [6] Ettayebi, M.; Prasad, S.M.; Morgan, E.A. arrest, it remains to be elucidated whether production of all Chloramphenicol-erythromycin resistance mutations in proteins is inhibited to the same extent or if translation of a 23S rRNA gene of Escherichia coli. J. Bacteriol. 1985, some cistrons is more sensitive to macrolide inhibition than 162, 551-557. the others. The importance of the inhibition of ribosome assembly is yet another parameter which must be studied in [7] Xiong, L.; Shah, S.; Mauvais, P.; Mankin, A.S. A ketolide more detail. resistance mutation in domain II of 23S rRNA reveals proximity of hairpin 35 to the peptidyl transferase centre. Mol. Microbiol. 1999, 31, 633-639. The mechanism of ribosome stall during induction of erm resistance and its relation to peptide-mediated resistance [8] Vester, B.; Garrett, R.A. A plasmid-coded and site- is unclear and thus, will require attention. Modeling or directed mutation in Escherichia coli 23S RNA that direct crystallographic studies of short nascent peptides on confers resistance to erythromycin: implications for the the ribosome may provide a useful structural background to mechanism of action of erythromycin. Biochimie 1987, approach these problems. It is obvious that detailed 69, 891-900. knowledge of the correlation between the structures of [9] Hansen, L.H.; Mauvais, P.; Douthwaite, S. The macrolide- inducing macrolides and the sequences of the nascent ketolide antibiotic binding site is formed by structures peptides mediating ribosome stall will be essential. in domains II and V of 23S ribosomal RNA. Mol. Microbiol. 1999, 31, 623-632. Although no clinical cases of peptide-mediated macrolide resistance have been reported to date, this type of resistance [10] Ban, N.; Nissen, P.; Hansen, J.; Moore, P.B.; Steitz, T.A. can be easily selected in the laboratory setting [56,60], and The complete atomic structure of the large ribosomal thus may potentially contribute to the overall resistance of a subunit at 2.4 A resolution. Science 2000, 289, 905-920. clinical isolate to macrolide antibiotics. Bacterial genomes [11] Harms, J.; Schluenzen, F.; Zarivach, R.; Bashan, A.; Gat, contain multiple short open reading frames, some encoding S.; Agmon, I.; Bartels, H.; Franceschi, F.; Yonath, A. High peptide sequences, which can potentially confer macrolide resolution structure of the large ribosomal subunit from resistance. Expression of such ORFs may easily avoid a mesophilic eubacterium. Cell 2001, 107, 679-688. detection. Thus a more focused investigation is required to evaluate the clinical importance of this resistance [12] Schlunzen, F.; Zarivach, R.; Harms, J.; Bashan, A.; Tocilj, mechanism. A.; Albrecht, R.; Yonath, A.; Franceschi, F. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 2001, 413, 814- 821. ACKNOWLEDGEMENTS [13] Hansen, J.; Ippolito, J.A.; Ban, N.; Nissen, P.; Moore, We thank Joyce Sutcliffe (Rib-X), Francois Franceschi P.B.; Steitz, T.A. The structures of four macrolide (Rib-X) and Frank Schlünzen (Max-Planck Research Unit for antibiotics bound to the large ribosomal subunit. Mol. Ribosomal Structure) for valuable advice and discussion, Cell 2002, 10, 117-128. Tanel Tenson (Tartu University) and Ada Yonath (Weizmann Institute) for communicating results prior to publication, [14] Garza-Ramos, G.; Xiong, L.; Zhong, P.; Mankin, A. Binding site of macrolide antibiotics on the ribosome: Joerg Harms (Max-Planck Research Unit for Ribosomal new resistance mutation identifies a specific interaction Structure) for preparing Fig. (2). The work was supported by of ketolides with rRNA. J. Bacteriol. 2002, 183, 6898- a grant from the National Institutes of Health to A.S.M. 6907. (GM 53762). [15] Depardieu, F.; Courvalin, P. Mutation in 23S rRNA responsible for resistance to 16-membered macrolides REFERENCES and streptogramins in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 2001, 45, 319-323. [1] Vazquez,D. Inhibitors of protein biosynthesis. Springer- Verlag: Berlin, Heidelberg, New York, 1979. [16] Douthwaite, S.; Hansen, L.H.; Mauvais, P. Macrolide- ketolide inhibition of MLS-resistant ribosomes is [2] Weisblum, B. Erythromycin resistance by ribosome improved by alternative drug interaction with domain II modification. Antimicrob. Agents Chemother. 1995, 39, of 23S rRNA. Mol. Microbiol. 2000, 36, 183-193. 577-585. [17] Capobianco, J.O.; Cao, Z.S.; Shortridge, V.D.; Ma, Z.K.; Flamm, R.K.; Zhong, P. Studies of the novel ketolide Macrolide Antibiotics: Binding Site, Mechanism of Action, Resistance Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 11

ABT-773: Transport, binding to ribosomes, and cryo-electron microscopy of the E. coli ribosome. Nature inhibition of protein synthesis in Streptococcus 1995, 376, 441-444. pneumoniae. Antimicrob. Agents Chemother. 2000, 44, 1562-1567. [33] Gabashvili, I.S.; Gregory, S.T.; Valle, M.; Grassucci, R.; Worbs, M.; Wahl, M.C.; Dahlberg, A.E.; Frank, J. The [18] Liu, M.; Kirpekar, F.; Van Wezel, G.P.; Douthwaite, S. The polypeptide tunel system in the ribosome and its gating tylosin resistance gene tlrB of Streptomyces fradiae in erythromycin resistance mutants of L4 and L22. EMBO encodes a methyltransferase that targets G748 in 23S J. 2001, 8, 181-188. rRNA. Mol. Microbiol. 2000, 37, 811-820. [34] Nakatogawa, H.; Ito, K. The ribosomal exit tunnel [19] Chittum, H.S.; Champney, W.S. Ribosomal protein gene functions as a discriminating gate. Cell 2002, 108, 629- sequence changes in erythromycin-resistant mutants of 636. Escherichia coli. J. Bacteriol. 1994, 176, 6192-6198. [35] Gong, F.; Yanofsky, C. Instruction of translating [20] Pardo, D.; Rosset, R. Properties of ribosomes from ribosome by nascent peptide. Science 2002, 297, 1864- erythromycin resistant mutants of Escherichia coli. Mol. 1867. Gen. Genet. 1977, 156, 267-271. [36] Tenson, T.; Ehrenberg, M. Regulatory nascent peptides [21] Spahn, C.M.T.; Prescott, C.D. Throwing a spanner in the in the ribosomal tunnel. Cell 2002, 108, 591-594. works: Antibiotics and the translation apparatus. J. Mol. Med. 1996, 74, 423-439. [37] Menninger, J.R. Mechanism of inhibition of protein synthesis by macrolide and lincosamide antibiotics. J. [22] Gregory, S.T.; Dahlberg, A.E. Erythromycin resistance Basic Clin. Phys. Pharmacol. 1995, 6, 229-250. mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA. J. Mol. [38] Chittum, H.S.; Champney, W.S. Erythromycin inhibits Biol. 1999, 289, 827-834. the assembly of the large ribosomal subunit in growing Escherichia coli cells. Curr. Microbiol. 1995, 30, 273- [23] Mao, J.C.-H.; Robishaw, E.E. Erythromycin, a 279. peptidyltransferase effector. Biochem. 1972, 11, 4864- 4872. [39] Champney, W.S.; Tober, C.L.; Burdine, R. A comparison of the inhibition of translation and 50S ribosomal [24] Vazquez, D. The Macrolide Antibiotics. In Antibiotics III. subunit formation in Staphylococcus aureus cells by Mechanism of action of antimicrobial and antitumor nine different macrolide antibiotics. Curr. Microbiol. agents; Corcoran,J.W.; Hahn, F.E. Eds.; Springer-Verlag: 1998, 37, 412-417. New York, Heidelberg, Berlin, 1975, pp. 459-479. [40] Usary, J.; Champney, W.S. Erythromycin inhibition of [25] Menninger, J.R.; Otto, D.P. Erythromycin, carbomycin, 50S ribosomal subunit formation in Escherichia coli and spiramycin inhibit protein synthesis by stimulating cells. Mol. Microbiol. 2001, 40, 951-962. the dissociation of peptidyl-tRNA from ribosomes. Antimicrob. Agents Chemother. 1982, 21, 810-818. [41] Champney, W.S.; Tober, C.L. Inhibition of translation and 50S ribosomal subunit formation in Staphylococcus [26] Champney, W.S.; Burdine, R. Macrolide antibiotics aureus cells by 11 different ketolide antibiotics. Curr. inhibit 50S ribosomal subunit assembly in Bacillus Microbiol. 1998, 37, 418-425. subtilis and Staphylococcus aureus. Antimicrob. Agents Chemother. 1995, 39, 2141-2144. [42] Alix, J.H.; Guerin, M.F. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli. Proc. [27] Poulsen, S.M.; Kofoed, C.; Vester, B. Inhibition of the Natl. Acad. Sci. USA 1993, 90, 9725-9729. ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and [43] Maki, J.A.; Schnobrich, D.J.; Culver, G.M. The DnaK tylosin. J. Mol. Biol. 2000, 304, 471-481. chaperone system facilitates 30S ribosomal subunit assembly. Mol. Cell 2002, 10, 129-138. [28] Mao, J.C.-H.; Robishaw, E.E. Effects of macrolides on peptide-bond formation and translocation. Biochem. [44] Tait-Kamradt, A.; Davies, T.; Appelbaum, P.C.; 1971, 10, 2054-2061. Depardieu, F.; Courvalin, P.; Petitpas, J.; Wondrack, L.; Walker, A.; Jacobs, M.R.; Sutcliffe, J. Two new [29] Tanaka, S.; Otaka, T.; Kaji, A. Furhter studies on the mechanisms of macrolide resistance in clinical strains of mechanism of erythromycin action. Biochim. Biophys. Streptococcus pneumoniae from Eastern Europe and Acta 1973, 331, 128-140. North America. Antimicrob. Agents Chemother. 2002, 44, 3395-3401. [30] Bernabeu, C.; Lake, J.A. Nascent polypeptide chains emerge from the exit domain of the large ribosomal [45] Cundliffe, E. How antibiotic-producing organisms avoid subunit: immune mapping of the nascent chain. Proc. suicide. Annu. Rev. Microbiol. 1989, 43, 207-233. Natl. Acad. Sci. USA 1982, 79, 3111-3115. [46] Hansen, J.L.; Schmeing, T.M.; Moore, P.B.; Steitz, T.A. [31] Yonath, A.; Leonard, K.R.; Wittmann, H.G. A tunnel in Structural insights into peptide bond formation. Proc. the large ribosomal subunit revealed by three- Natl. Acad. Sci. USA 2002, 99, 11670-11675. dimensional image reconstruction. Science 1987, 236, 813-816. [47] Skinner, R.; Cundliffe, E.; Schmidt, F.J. Site of action of a ribosomal RNA methylase responsible for resistance to [32] Frank, J.; Zhu, J.; Penczek, P.; Li, Y.; Srivastava, S.; erythromycin and other antibiotics. J. Biol. Chem. 1983, Verschoor, A.; Radermacher, M.; Grassucci, R.; Lata, R.K.; 258, 12702-12706. Agrawal, R.K. A model of protein synthesis based on 12 Current Topics in Medicinal Chemistry, 2003, Vol. 3, No. 4 Gaynor and Mankin

[48] Rosato, A.; Vicarini, H.; Leclercq, R. Inducible or of induction by macrolide-lincosamide-streptogramin B constitutive expression of resistance in clinical isolates antibiotics. J. Bacteriol. 1990, 172, 3772-3779. of streptococci and enterococci cross-resistant to erythromycin and lincomycin. J. Antimicrob. Chemother. [56] Tenson, T.; DeBlasio, A.; Mankin, A. A functional 1999, 43, 559-562. peptide encoded in the Escherichia coli 23S rRNA. Proc. Natl. Acad. Sci. USA 1996, 93, 5641-5646. [49] Giovanetti, E.; Montanari, M.P.; Mingoia, M.; Varaldo, P.E. Phenotypes and genotypes of erythromycin- [57] Tenson, T.; Xiong, L.; Kloss, P.; Mankin, A.S. resistant Streptococcus pyogenes strains in Italy and Erythromycin resistance peptides selected from random heterogeneity of inducibly resistant strains. Antimicrob. peptide libraries. J. Biol. Chem. 1997, 272, 17425- Agents Chemother. 1999, 43, 1935-1940. 17430. [50] Dubnau, D. Induction of ermC requires translation of the [58] Tripathi, S.; Kloss, P.S.; Mankin, A.S. Ketolide resistance leader peptide. EMBO J. 1985, 4, 533-537. conferred by short peptides. J. Biol. Chem. 1998, 273, 20073-20077. [51] Weisblum, B. Macrolide resistance. Drug Resist. Updates 1998, 1, 29-41. [59] Heurgué-Hamard, V.; Karimi, R.; Mora, L.; MacDougall, J.; Leboeuf, C.; Grentzmann, G.; Ehrenberg, M.; [52] Gryczan, T.J.; Grandi, G.; Hahn, J.; Grandi, R.; Dubnau, D. Buckingham, R.H. Ribosome release factor RF4 and Conformational alteration of mRNA structure and the termination factor RF3 are involved in dissociation of posttranscriptional regulation of erythromycin-induced peptidyl-tRNA from the ribosome. EMBO J. 1998, 17, drug resistance. Nucleic Acids Res. 1980, 8, 6081-6097. 808-816. [53] Mayford, M.; Weisblum, B. Conformational alterations [60] Douthwaite, S.; Prince, J.B.; Noller, H.F. Evidence for in the ermC transcript in vivo during induction. EMBO J. functional interaction between domains II and V of 23S 1989, 8, 4307-4314. ribosomal RNA from erythromycin resistant mutant. Proc. Natl. Acad. Sci. USA 1985, 82, 8330-8334. [54] Zhong, P.; Cao, Z.; Hammond, R.; Chen, Y.; Beyer, J.; Shortridge, V.D.; Phan, L.Y.; Pratt, S.; Capobianco, J.; [61] Yusupov, M.M.; Yusupova, G.Z.; Baucom, A.; Lieberman, Reich, K.A.; Flamm, R.K.; Or, Y.S.; Katz, L. Induction of K.; Earnest, T.N.; Cate, J.H.; Noller, H.F. Crystal structure ribosome methylation in MLS-resistant Streptococcus of the ribosome at 5.5 A resolution. Science 2001, 292, pneumoniae by macrolides and ketolides. Microb. Drug 883-896. Resist. 1999, 5, 183-188. [62] Carson, M. Ribbons. Methods Enzymol. 1997, 277, 493- [55] Mayford, M.; Weisblum, B. The ermC leader peptide: 505. amino acid alterations leading to differential efficiency