Peptides 22 (2001) 1661–1668
Review
Short peptides conferring resistance to macrolide antibiotics Tanel Tensona, Alexander S. Mankinb,* a
b
Institute of Molecular and Cell Biology, Tartu University, Riia 23, Tartu 51010, Estonia Center for Pharmaceutical Biotechnology - m/c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607, USA Received 22 December 2000; accepted 17 April 2001
Abstract Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction 1.1. Macrolide antibiotics: binding site and mechanism of action All proteins in the cell, large or small, are synthesized by the ribosome. The ribosome consists of two subunits. The small ribosomal subunit is engaged in decoding of genetic information, while the large subunit assembles amino acids into a polypeptide chain. Catalysis of peptide bond formation and amino acid polymerization occurs in the peptidyl transferase center located on the large subunit. At a short distance form the peptidyl transferase center is the entrance to the nascent peptide channel through which a newly synthesized polypeptide leaves the ribosome. Many natural and synthetic antibiotics inhibit bacterial growth by interfering with protein synthesis. Most of these drugs act upon the ribosome. Among the ribosome-targeted antibiotics, one of the most important groups is the 14member ring macrolides [1]. Macrolides bind to the large ribosomal subunit in the vicinity of the peptidyl transferase center (see [37–39] for review). However, in contrast to many other drugs that act upon the large subunit, macrolides do not inhibit catalytic activity of ribosomal peptidyl trans* Corresponding author. Tel.: ⫹1-312-413-1406; fax: ⫹1-312-4139304. E-mail addresses:
[email protected] (A. Mankin),
[email protected] (T. Tenson).
ferase directly. Instead, they interfere with the growth of the nascent peptide chain during early rounds of translation [35,36]. Though the exact mechanism of such inhibition is not clear, two (not necessarily exclusive) models have emerged. In one commonly accepted scenario, macrolides barricade the entrance to the nascent peptide channel, thus preventing growth of the newly synthesized peptide [35]. On the other hand, macrolides were shown to compete with the peptidyl tRNA for binding to the ribosome and therefore can cause spontaneous dissociation of the peptidyl-tRNA before the nascent peptide enters the exit channel [19]. In either of these scenarios, the macrolide molecule is expected to interact with the growing polypeptide chain a few amino acids away from the peptidyl transferase center. The central component of macrolide chemical structure is a lactone ring carrying a number of substitutions (Fig. 1). The first clinically important drug of this group of antibiotics, and also the best known, is erythromycin, which represents the first generation of 14-member ring macrolides. Macrolides of the second generation, such as clarithromycin or roxithromycin, are characterized by better stability and improved spectrum of activity. Subsequent rapid spread of antibiotic-resistant strains has stimulated the search for novel derivatives. Macrolides of the third generation, called ketolides, contain a keto group instead of the cladinose residue at position 3 of the lactone ring and carry alkyl-aryl side chains. Ketolides not only show an improved activity profile compared to the drugs of the second gener-
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1.3. rRNA-encoded E-peptide
Fig. 1. Chemical structures of macrolide antibiotics of the first (erythromycin), second (clarithromycin) and third (telithromycin) generation. These and other macrolides were used to isolate and characterize peptides conferring macrolide resistance.
ation, but are also more active against certain macrolideresistant strains [1,40]. 1.2. Main mechanisms of macrolide resistance The binding site of 14-member ring macrolides includes two distinct segments of the 23S rRNA located in close vicinity in the ribosome tertiary structure. The best characterized rRNA site involved in macrolide binding is a segment of the so called central loop of domain V, in which positions 2058 and 2059 (E. coli numbering) are critical for the drug binding [20]. The main mechanism of macrolide resistance is based on a modification of the drug binding site in the ribosome, specifically, methylation of A2058 by specific methyl transferase enzymes [16,25]. Methylation of A2058 results in a dramatic reduction of drug binding [10]. rRNA mutations in the vicinity of A2058 and also in helix 35 of domain II, another rRNA segment involved in macrolide binding, can also render cells resistant to the macrolide action [14,42]. Not only mutations in rRNA, but also mutations in ribosomal proteins L4 and L22 confer resistance to macrolides [2,8,29]. Analysis of the protein mutations suggests that they may cause allosteric changes in the conformation of rRNA in the macrolide binding site [13]. The second most important mechanism of cell resistance to macrolides is based on antibiotic efflux (27,28, see also 26,40 for review). Specialized or broad-spectrum drug-efflux transporters can efficiently reduce the intracellular drug concentration and render cells drug resistant. Resistance mechanisms based on enzymatic chemical modification of the drug which were described for several isolates do not play a major role in clinical macrolide resistance [3]. A novel mechanism of macrolide resistance, based on the expression of a specific short peptide in the cell, was discovered in our laboratories several years ago [30,31]. In this paper, we review previous data and more recent findings, which provide insights into this mechanism of drug resistance. We believe that understanding how short peptides can render cells resistant to macrolides will not only explain this particular resistance mechanism, but will also provide important clues to the mode of macrolide action and basic mechanisms of protein synthesis.
1.3.1. The first encounter The phenomenon of peptide-mediated macrolide resistance was first discovered coincidentally in an experiment where fragments of rRNA were expressed in vivo in search for fragments that can bind antibiotics [30]. E. coli cells expressing random fragments of rrnB operon were screened for the presence of erythromycin (Ery) resistant clones. Several Eryr clones were identified. In all such clones, resistance depended on expression of rRNA fragments all of which encompassed the region 1233–1348 in 23S rRNA. The rRNA fragments expressed in these clones were designated E-RNA for erythromycin resistance RNA. The smallest of the E-RNAs in the originally selected Eryr clones contained a 116 nucleotide long segment of the 23S rRNA. Subsequent deletion analysis showed that expression of an rRNA fragment only 34 nucleotides long (E-RNA34), corresponding to the E. coli 23S rRNA sequence 1235–1268, was sufficient to render cells resistant to erythromycin. 1.3.2. rRNA or mRNA? Neither biochemical nor genetic data implicated the segment of 23S RNA corresponding to E-RNA34 in erythromycin binding, suggesting a completely new mode of drug resistance conferred by its expression. Mutational analysis of E-RNA34 revealed three regions important for the Ery resistance: the 5⬘-terminal five nucleotides, GGAGG, a trinucleotide GUG and yet another tri-nucleotide UAA (Fig. 2A) [30]. Remarkably, these three segments correspond to the Shine-Dalgarno sequence, initiator and terminator codons of a short open reading frame (ORF) which codes for a pentapeptide MRMLT. Subsequent biochemical and genetic analysis showed that translation of this mini-gene is required and sufficient to render cells resistant to low concentrations of erythromycin. E-RNA34 with its pentapeptide mini-gene is derived from 23S rRNA. Does it mean that wild-type E. coli cells are constitutively resistant to erythromycin due to expression of the rRNA-encoded pentapeptide? Probably not. It is unlikely that significant amounts of the rRNA-encoded pentapeptide can be expressed in the cell under “normal” conditions. Not only is 23S rRNA associated with ribosomal proteins and can hardly be directly translated, but also the Shine-Dalgarno region of the mini-gene is sequestered in the 23S rRNA secondary structure (Fig. 2B) - thus making its translation impossible. All E-RNAs expressed in the Eryr clones selected from the random rRNA fragment library lacked the 5⬘ half of the hairpin sequestering the ShineDalgarno region of the mini-gene and therefore, peptide expression was not hampered. Expression of the rRNAencoded pentapeptide can be activated by specific mutations. A spontaneous deletion of 12 nucleotides (positions 1219 –1230) from the 23S rRNA gene was shown to cause resistance to erythromycin [11,12]. Such deletion evidently destabilized the hairpin, making the ribosome-binding site
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peptide was added to the cell-free translation system, inhibitory action of erythromycin was diminished. In this experiment, ribosomes were allowed to synthesize the MRMLT peptide by translating E-RNA34 and then were “re-programmed” with MS2 RNA. The ribosomes that had a chance to translate E-RNA34 prior to translation of MS2 RNA became tolerant to erythromycin. This result suggested that a ribosome becomes erythromycin resistant only if the MRMLT peptide was synthesized on that very same ribosome (in the other words, that the peptide acts in cis). The important implication of this finding was that the ribosome is the primary site and possibly the target of action of the rRNA-encoded MRMLT peptide.
Fig. 2. A. Nucleotide sequence of an E. coli 23S rRNA fragment encoding the E-peptide mini-gene. Nucleotide sequences whose mutations eliminate peptide-mediated resistance are underlined. Amino acid sequence of the encoded E-peptide is shown beneath the corresponding codons of the mini-gene. B. Secondary structure of the segment of E. coli 23S rRNA encompassing the E-peptide mini-gene. The mini-gene and its ribosome binding site are shown in bold and the Shine-Dalgarno sequence of the mini-gene is boxed. E. coli 23S rRNA numeration is shown.
of the E-peptide mini-gene more accessible, thus activating E-peptide expression. Other deletion and nucleotide substitutions in this hairpin can activate mini-gene expression through a similar mechanism [9]. 1.3.3. How does the peptide work? How can a short peptide with a molecular weight comparable to that of erythromycin render the bacterial cell resistant to the drug? One of the plausible models was that the peptide could bind erythromycin and sequester it in an inactive complex. To test this model, activity of a synthetic pentapeptide was tested in a cell-free translation system. In vitro translation of phage MS2 RNA is efficiently inhibited by low concentrations of erythromycin. Contrary to expectations, addition of excess of the synthetic MRMLT peptide (up to 1 mM) to the cell-free translation system did not alleviate inhibitory action of erythromycin, therefore ruling out direct sequestering of the drug by the peptide [30]. In contrast, when E-RNA34 instead of the synthetic
1.3.4. Peptide gene in rRNA: why is it there? The idea that rRNA in addition to its structural and functional role in the ribosome could code for proteins has been around for a long time. A few relatively long open reading frames have been identified in rRNA [5,6,23]. Nevertheless, the translation products of these ORFs were never detected and functions of the putative proteins were never recognized. Finding of the E-peptide raises the question about translation of ribosomal RNA again. Because of the association of 23S rRNA with ribosomal proteins and because of its secondary structure, the MRMLT peptide mini-gene is not expressed unless mutations occur in the 23S rRNA gene or 23S rRNA undergoes site-specific fragmentation. Such fragmentation can potentially occur under specific physiological conditions. However, so far, we were unable to identify the conditions that would lead to the expression of the rRNA-encoded peptide in the cell. In general, it remains unclear if the presence of the peptide mini-gene in rRNA has a biological purpose (in the other words, whether it is a result of evolutionary selection or whether it is simply coincidental). The mini-gene is well conserved among different branches of eubacteria, missing only in the Rhodobacter-Flexibacter branch. This conservation argues in favor of functional importance of the rRNAencoded mini-gene. Nevertheless, its functions remain obscure. We can only speculate what the possible physiological significance of an rRNA-encoded peptide could be. The cis mode of peptide action in the macrolide resistance mechanism suggests that the peptide can possibly modulate properties of the ribosome. However, while expression of some other short peptides have profound effects on protein synthesis and cell growth [15,32], overexpression of the rRNA-encoded E-peptide did not significantly change growth characteristics of the cell. If the peptide mini-gene is present in rRNA for a reason, it is certainly not to render cells resistant to erythromycin. Rather, resistance to the drug is a consequence of some special mode of interaction between the peptide and the ribosome (see below).
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onstrated that any mutations eliminating the stop codon of the pentapeptide mini-gene abolished erythromycin resistance [30]. Therefore, the mere presence of an E-peptide sequence at the N-terminus of a longer polypeptide could not apparently render ribosomes resistant to erythromycin. The result of screening a 21-codon library confirmed this observation and also showed that presence of the E-peptide sequence at the C-terminus of a polypeptide could not confer erythromycin resistance. Thus, an erythromycin resistance peptide cannot be part of a longer protein and the small size of the peptide is critical for its activity. The small size of the peptide can be important if termination of translation plays a role in the mechanism of the peptide action. Fig. 3. Plasmid library in pPOT1AE vector [33] used for selection of peptides conferring resistance to macrolides. Promoter (Ptac) and terminator (Ttrp) of the mini-gene are shown by black boxes. Shine-Dalgarno sequence, initiator and terminator codons of the mini-gene are underlined. The pentapeptide library contained 4 random codons (12 random nucleotide positions) while the 21-codon library contained 20 random codons (60 random nucleotide positions).
1.4. Mini-gene libraries as a tool for selection of functional peptides Mutational analysis of the rRNA-encoded mini-gene suggested that peptides with sequences other than MRMLT can render cells resistant to erythromycin. In order to clarify which properties allow a peptide to confer resistance, a variety of erythromycin resistance peptides (E-peptides) were selected from random mini-gene libraries (Fig. 3) [33]. In such libraries, each individual bacterial clone expresses a peptide encoded in a specific mini-gene. Two libraries were used to select peptides conferring resistance to erythromycin. One contained 5-codon-long mini-genes (4 of them randomized). The other contained 21-codon-long minigenes (20 of them randomized). Comparison of peptides expressed in erythromycin resistant clones revealed sequence and size requirements for the peptide activity. 1.4.1. Size is critical for activity of E-peptides There are three termination codon in the genetic code. Therefore, about 2/3 of the clones in the 21-codon library are expected to have in-frame stop codons. Because of this, the library provides an excellent tool for investigating the size requirement for functional peptides. As expected, a broad distribution of sizes of the encoded peptides were found in unselected, randomly picked clones. In contrast, the majority of the peptides expressed in Eryr clones fell within an amazingly narrow size range. Eleven out of twelve peptides were four, five or six amino acids long, suggesting that the optimal length of erythromycin resistance peptides should be within this range [33]. In agreement with this conclusion, the originally described rRNAencoded peptide (MRMLT) was five amino acids long. Genetic analysis of the rRNA-encoded mini-gene dem-
1.4.2. E-peptide consensus sequence If analysis of clones isolated from the 21-codon library revealed peptide size requirements, then screening the 5-codon library provided information regarding the sequence of the active E-peptides. Comparison of sequences of 52 pentapeptide mini-genes found in Eryr clones showed a strong tendency for having Leu or Ile in the third position and a hydrophobic amino acid (most commonly Val) in the C-terminal position. Of sixteen peptides that conferred resistance to high concentrations of erythromycin (1 mg/ml), only one lacked Leu or Ile in the third position, and only one peptide did not have a hydrophobic amino acid in the 5th position [33] (Fig. 4A). Though all the resistance peptides were selected in E. coli system, their activity is not limited to this organism because at least some of the selected peptides could confer erythromycin resistance in evolutionary distant Bacillus subtilis and Proteus mirabilis [22]. 1.5. Different peptides confer resistance to different macrolides E-peptide did not affect cell sensitivity to chloramphenicol and clindamycin, structurally different antibiotics which compete with erythromycin for binding to the ribosome [7]. However, E-peptide rendered cells resistant to low concentrations of oleandomycin and spiramycin, which, together with erythromycin, belong to the family of macrolide antibiotics. This observation raised the possibility that E-peptides could render cells resistant to all types of macrolides. Alternatively, different peptides could be required to confer resistance to different macrolide antibiotics. Selection of resistance peptides using a variety of macrolide antibiotics confirmed the latter model [34] (Fig. 4). Two general groups of resistance peptides emerged from these experiments. The first group consisted of peptides that render cells resistant to macrolide antibiotics containing a cladinose residue at position 3 of the lactone ring (C-peptides). C-peptides show a very strong bias in amino acid composition with a prevalence of the hydrophobic amino acid residues Leu, Ile, Val, Ala, Phe and Trp. The preponderance of hydrophobic amino acid residues is especially notable in the third and fifth position of the pentapeptide,
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Fig. 4. Nucleotide sequences of mini-genes and amino acid sequences of the encoded peptides conferring resistance to different macrolide antibiotics. A. C-peptides conferring resistance to cladinose-containing compounds, erythromycin, clarythromycin, roxithromycin and RU69874. B. K-peptides conferring resistance to ketolides, telithromycin, RU64399 and HMR3004. All the compounds used in selection experiments were obtained from Aventis-Pharma.
while the second and especially the fourth position exhibit higher amino acid diversity. The most conserved position in C-peptides is the third, which is most commonly occupied by leucine. Peptides of the second group (K-peptides) confer resistance to ketolides - antibiotics which possess keto function in place of the cladinose residue. K-peptides are less hydrophobic than C-peptides. They contain positively charged Arg or Lys in the second or sometimes fourth position, but lack Leu in the third position. The relationship between the drug structure and sequence of the resistance peptides is especially evident when compounds differing from each other only by the presence or absence of cladinose moiety are compared (for example, RU69874 and telithromycin). This example clearly shows that structurally distinct peptides are required to confer resistance to the drugs with only a small difference in their molecular structures. Other variations in the drug structure also call for different consensus sequences of the resistance peptides (Tenson and Mankin, unpublished results). 1.6. Brushing the ribosome The most straightforward explanation of the correlation between the sequence of the resistance peptides and the structure of a macrolide antibiotic is that at some point, a peptide will interact directly with the drug molecule. The cell-free experiments with the rRNA-encoded MRMLT peptide (discussed above) showed that E-peptides do not bind the drug “outside” the ribosome. Thus, we arrive at the conclusion that peptide-drug interaction must take place in the ribosome. Crystallographic structure of the large ribosomal subunit showed that extended segments of ribosomal proteins L4 and L22 form a narrow “gate” located at the entrance of the nascent peptide channel [4,21]. Biochemical and genetic data indicate that macrolides bind very close to this gate. This is the most probable site where the interaction between the drug and the resistance peptide occurs. The distance
between the peptidyl transferase center and macrolide binding site is approximately 14Å. Therefore, when the C- or K-peptides synthesized by the ribosome reach the size of 4 –5 amino acids, they can come in direct contact with the macrolide molecule. Genetic and biochemical studies revealed several important facts pertaining to the mode of action of the resistance peptides. 1) Resistance peptides are small, 4-to-6 amino acids long. 2) There is a correlation between the sequence of the resistance peptides and the structure of the drug to which the peptide confers resistance. 3) Peptides act in cis: only the ribosome on which the peptide was synthesized becomes resistant to the drug. 4) Free peptides do not bind to the antibiotic but may interact with the drug on the ribosome. All these data can be accounted for by a “bottle brush” model of the peptide action [34] (Fig. 5). Macrolides do not block initiation of translation or the first several rounds of amino acid polymerization. Therefore, the ribosome with the bound macrolide antibiotic can still translate the resistance peptide mini-gene. When synthesis of the peptide is almost finished (when the nascent peptide is 4 –5 amino acid residues long), the N-terminal segment of the peptide reaches the site where the antibiotic is bound and the peptide forms specific contacts with the drug molecule. During termination of peptide synthesis when the peptide is
Fig. 5. “Bottle brush” model of the resistance peptide action. Translation of a resistance peptide (one of the C-peptides is shown) may remove antibiotic from its binding site on the ribosome. This will free the ribosome for translation of a cellular protein. The cumulative effect of translating the mini-genes encoding resistance peptides will be an overall increase in the population of drug-free ribosomes in the cell.
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released from the peptidyl-tRNA, or during the last translocation event, the peptide “kicks” the drug out of its binding site thus removing antibiotic from the ribosome. Once the antibiotic is removed, the ribosome can engage in translation of a cellular protein. If it has enough time to polymerize the first 4 – 6 amino acids before a macrolide molecule binds again to the ribosome, then the drug binding site will be occupied by the nascent polypeptide and the ribosome can successfully finish translation of the protein. After the release of the completed protein, the ribosome can either initiate translation of a new protein or it can be invaded by an antibiotic and remain inactive until it is again “cleaned” by translation of a resistance peptide. Frequent translation of resistance peptide mini-genes essentially increases the population of drug-free ribosomes in the cell. At a relatively low drug concentration this is sufficient for the cell to continue its growth. 1.7. Resistance peptides and inducible resistance The bottle brush model suggests direct interaction of the growing polypeptide chain with the macrolide molecule on the ribosome. Such interaction is reminiscent of that implicated in the induction of Erm genes (see [38] for review). Erm genes code for methyltransferase enzymes that render cells resistant to macrolides (as well as streptogramins and lincosamides) by methylating a specific adenine residue (A2058) located in the drug binding site in 23S rRNA. Induction of erm expression involves conformational isomerization of its mRNA into a translationally active form. The leader region of erm mRNA that precedes the erm cistron contains a short open reading frame. In the absence of antibiotic, mRNA assumes a conformation where the translation initiation region of erm is sequestered in a hairpin structure, which precludes erm expression (Fig. 6). In the presence of low concentrations of an inducing macrolide antibiotic (for example, erythromycin), the ribosome stalls on the leader ORF. This switches the mRNA secondary structure so that erm translation becomes possible. Stalling of the ribosome in the leader ORF occurs when the eighth codon (Val) of the leader ORF is positioned in the ribosomal P site [17]. The identity of Val8 is essential for stalling. Similarly, alterations of the two preceding amino acids, Ile6 and Phe7, strongly diminishes ribosome stalling and erm induction. The amino acid sequence of this leader peptide segment that is critical for ribosome stalling (Ile6 Phe7 - Val8) is remarkably similar to the consensus sequence of E-peptides. When synthesis of the E-peptide is completed, right before its translation is terminated, the last (most commonly Val) codon of E-peptide ORF is positioned in the ribosomal P-site. The semi-ultimate position of the E-peptide is frequently a hydrophobic amino acid and the preceding position is either leucine or isoleucine (Fig. 4). Analogy between erm leader peptides and E-peptides extends even further. While alteration of the E-peptide sequence changes the spectrum of antibiotics to which the peptide confers resistance, alterations in the erm leader
Fig. 6. Similarity between erm leader peptides and macrolide resistance peptides. A. “Inactive” conformations of the leader region of erm mRNA. Shine-Dalgarno regions and initiator codons of the leader peptide and Erm ORFs are underlined. Amino acid sequence of the leader peptide is shown above the corresponding codons of the leader ORF. B. “Active” conformation of erm mRNA. Stalling of the ribosome occurs when the eighth (Val) codon of the leader ORF is positioned in the ribosomal P-site. Switching of mRNA conformation activates translation of the erm cistron. C. Pre-termination complex of the ribosome with mRNA of the C-peptide MVLFV that confers resistance to erythromycin.
peptide affects the spectrum of antibiotics which cause ribosome stalling and erm induction [18]. This indicates that not only resistance peptides but also erm leader peptides interact with the drug on the ribosome. The effect of such interaction is however, strikingly different: possible removal of antibiotic in case of resistance peptide and ribosome stalling in case of erm leader peptides. Such disparity in the mode of peptide action can be related to a difference in the length of the nascent peptides. Termination of the translation of the five amino acid long nascent E-peptide may lead to antibiotic expulsion, while a longer nascent peptide of the erm leader ORF may get “stuck” in the ribosome in the presence of the drug. Alternatively, the difference in ribosome response can depend on the different N-terminal sequences of the E- and leader ORF peptides.
2. Conclusions and future directions Expression of C- or K- peptides renders cells resistant to relatively low concentrations of macrolide antibiotics. Therefore, this mechanism can not play a major role in the
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drug resistance of clinical bacterial strains. Nevertheless, it can contribute to the initial survival rate of the pathogenic bacteria during antibiotic treatment and therefore increase the chances of acquiring one of the major resistance mechanisms. A spontaneous 12 bp deletion in 23S rRNA gene results in the expression of the E-peptide mini-gene encoded in bacterial rRNA [12]. There are many other potential mini-ORFs present in the bacterial genomes which can code for resistance peptides. A single mutational event can generate an appropriate promoter or translation initiation site for such an ORF and activate peptide expression. Apart from its potential clinical significance, the phenomenon of peptide-mediated macrolide resistance is important because it reveals a new aspect of interaction of nascent peptide with the ribosome and macrolide antibiotics. The correlation between the peptide structure and chemical structure of the drug suggests direct peptide-drug contact on the ribosome. However, the detailed mechanism of such interaction remains obscure. It is possible that the peptide structure needs to be constrained by the ribosome to allow specific contacts with the drug, but it has never been proven. Other questions closely related to the activity of Cand K-peptides and the mode of macrolide action also await exploration. What is the exact size of the nascent peptide when macrolides start to exert their inhibitory action? Does this length depend on the nascent peptide sequence? Is translation of all different polypeptides in the cell affected by macrolides to the same extent? The recently unveiled high resolution crystal structures of the ribosomal subunits are expected to bring our understanding of how the ribosome works to a new level [4,24, 41]. The next critical and long-awaited leap in our knowledge of the macrolide binding site will be brought about when crystal structures of ribosome-macrolide complexes become public. There is a hope that these structures, in combination with biochemical data, will help us to understand the intimate relations among the ribosome, nascent peptide and macrolide antibiotics.
Acknowledgments We thank Liqun Xiong and Marne Gaynor for their expert technical assistance and Maria Gomez for help in preparing the manuscript. This work was supported by National Institutes of Health grants GM53762 and TW00870 and Aventis Pharma Research Grant (to A.S.M.) and Estonian Science Foundation grant N 4443 (to T.T.).
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