Inhibition Of Peptide Bond Formation Research Articles

Chloramphenicol Interferes with 50S Ribosomal Subunit Maturation via Direct and Indirect Mechanisms.

Chloramphenicol (CAM), a well-known broad-spectrum antibiotic, inhibits peptide bond formation in bacterial ribosomes. It has been reported to affect ribosome assembly mainly through disrupting the balance of ribosomal proteins. The present study investigates the multifaceted effects of CAM on the maturation of the 50S ribosomal subunit in Escherichia coli (E. coli). Using label-free quantitative mass spectrometry (LFQ-MS), we observed that CAM treatment also leads to the upregulation of assembly factors. Further cryo-electron microscopy (cryo-EM) analysis of the ribosomal precursors characterized the CAM-treatment-accumulated pre-50S intermediates. Heterogeneous reconstruction identified 26 distinct pre-50S intermediates, which were categorized into nine main states based on their structural features. Our structural analysis highlighted that CAM severely impedes the formation of the central protuberance (CP), H89, and H58 during 50S ribosomal subunit maturation. The ELISA assay further demonstrated the direct binding of CAM to the ribosomal precursors, suggesting that the interference with 50S maturation occurs through a combination of direct and indirect mechanisms. These findings provide new insights into the mechanism of the action of CAM and provide a foundation for a better understanding of the assembly landscapes of the ribosome.

Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs.

As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3' adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

A Safety Study of the Addition of Omacetaxine to the Standard-of-Care Induction Regimen of Cytarabine and Idarubicin in Newly-Diagnosed AML Patients

Background: Omacetaxine mepesuccinate (OM) is a semi-synthetic form of Homoharringtonine (HH), a cephalotaxine alkaloid. OM induces cell apoptosis by inhibiting peptide bond formation during mRNA translation, with rapid loss of short-lived proteins, such as MCL-1, c-MYC, and Cyclin D1 (Lu, J Hematol Oncol. 2014, 7: 2). Notably, cytarabine synergizes with HH in causing apoptosis of leukemia cells in vitro. A phase III RCT in China of 620 patients with de novo AML demonstrated superior CR and 3-yr survival rates upon addition of HH to a standard 2-drug AML induction therapy (‘7 + 3‘; Jin, Lancet Oncol. 2013, 14:599). Thus, we hypothesized that OM, at an appropriate dose, would similarly enhance the efficacy of a 7 + 3 regimen. OM is FDA-approved for the treatment of TKI-resistant CML. The MTD of 1.25 mg/m2/d SQ for 14 days every 28 days, as determined in a phase I/II CML trial of OM (Quintás-Cardama, Cancer 2007, 109: 248), served as a basis for the dose escalation used in this study.Methods: The primary endpoint of this phase I safety trial was to determine the optimally safe and active dose (OD) of OM when added to a standard 7 + 3 induction regimen, cytarabine and idarubicin. OM was administered SQ q12h d1-7 with cytarabine (100mg/m2 CIV) d1-7 and idarubicin (12mg/m2 IV) d1-3. Four dose levels were tested, starting with OM 0.625 mg/m2 q12h (further dose levels: 1.25, 2.0, 3.0, and 4.2 mg/m2 q12h). All newly diagnosed, untreated de novo or secondary AML patients, aged 18-70y with ECOG PS of 0-3 were eligible for this study. Secondary endpoints included overall response rate (ORR) and overall and event free survival (OS, EFS). Hematologic toxicity (HT) was defined as incomplete hematologic recovery; ANC < 1.0 x 109/L or platelet count < 100 x 109/L present at d49, with the bone marrow documented to be free of leukemic infiltration. Dose escalation was based on the EffTox design (Biometrics 2004, 60:684), a Bayesian adaptive design which considers the trade-off between efficacy and toxicity in determining the OD for Phase II trials.Results: Twenty-two patients, median age 58 (range 25-69) years were enrolled from June 2015 to June 2018. 12 patients (54.5%) had adverse cytogenetics, 6 (27%) intermediate risk, 3 (13.7%) favorable risk and 1 patient's cytogenetic risk was unknown (fibrotic BM). Eight patients demonstrated disease evolution from myelodysplastic syndrome (MDS). Altogether 16 of the 22 patients (73%) were deemed high risk based on cytogenetics or MDS-AML evolution. The EffTox design was implemented until cohort 4 (3 mg/m2 q12h), where 2 of 3 patients experienced a grade 5 non-hematologic toxicity (NHT), resulting in a dose-limiting toxicity (DLT). Since no DLTs were observed in cohort 3, an additional 5 patients were thus enrolled at this dose level to ensure safety. The OD was determined to be the dose level used in cohort 3: OM 2 mg/m2. No HTs were observed in 21 of 22 patients, (one patient not evaluable). The most common non-hematologic treatment emergent adverse events (TEAEs) of any grade were fever (68%), nausea (64%), vomiting (55%), hyperglycemia (41%), diarrhea (41%), mucositis (36%), headache (36%), sinus tachycardia (32%), rash/dermatitis (32%), and abdominal pain (32%). The most prevalent non-hematologic grade 3/4 TEAEs were febrile neutropenia (23%), hypoxia (18%), hyperglycemia (18%), and dyspnea (18%). ORR (CR and CRi) was 45.5%. Median OS was 605 days and EFS was 100 days.Conclusion: In this population with predominantly high-risk AML, the combination of OM with a standard 7 + 3 regimen demonstrates a manageable safety profile with acceptable efficacy. As ~ 25% of patients achieving CR with ‘7 + 3‘ do so after a second induction (based on meta-analysis of 6 trials, n = 1980, see Cancer 2010, 116: 5012), the ORR here is comparable to those receiving a single standard of care induction. The results in this high-risk group are therefore promising and warrant further investigation in a phase II trial. At present, we are assessing leukemic blast MCL protein expression in stored pre-treatment samples to determine if this predicts OM efficacy.NCT02440568. Teva has performed a Medical Accuracy Review of this abstract. DisclosuresKhan:Teva: Speakers Bureau. Patel:Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Honoraria.

MRNA-specific translation regulation by a ribosome-associated ncRNA in Haloferax volcanii

Regulation of gene expression at the translational level allows rapid adaptation of cellular proteomes to quickly changing environmental conditions and is thus central for prokaryotic organisms. Small non-coding RNAs (sRNAs) have been reported to effectively orchestrate translation control in bacteria and archaea mainly by targeting mRNAs by partial base complementarity. Here we report an unprecedented mechanism how sRNAs are capable of modulating protein biosynthesis in the halophilic archaeon Haloferax volcanii. By analyzing the ribosome-associated ncRNAs (rancRNAs) under different stress conditions we identified an intergenic sRNA, termed rancRNA_s194, that is primarily expressed during exponential growth under all tested conditions. By interaction with the ribosome rancRNA_s194 inhibits peptide bond formation and protein synthesis in vitro but appears to target a specific mRNA in vivo. The respective knock-out strain shows a reduced lag phase in media containing xylose as sole carbon source and outcompetes the wildtype cells under these conditions. Mass spectrometry, polysome profiling and mRNA binding competition experiments suggest that rancRNA_s194 prevents the cstA mRNA from being efficiently translated by H. volcanii ribosomes. These findings enlarge the regulatory repertoire of archaeal sRNAs in modulating post-transcriptional gene expression.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

ErmBL Translation on the Ribosome in the Presence of Erythromycin is Stalled by Inhibition of Peptide Bond Formation

Translation of the Streptococcus sanguis ErmBL peptide is stalled in the presence of the antibiotic erythromycin, which binds inside the ribosomal exit tunnel. The stalling regulates the expression of the downstream gene of the resistance methyltransferase ErmB. Here, we investigate the stalling mechanism by explicit-solvent all-atom molecular dynamics simulations of the ribsome-ErmBL complex in the presence and absence of erythromycin. The simulations, totaling 12 µs simulation time, were started from a 3.6-A-resolution cryo-EM structure. In the cryo-EM structure the ribose of A76 of the peptidyl tRNA is shifted from its canonical pre-attack position. In the simulations without erythromycin, the ErmBL peptide explores conformations, which would overlap with the antibiotic. Together, these two findings show that erythromycin actively restrains the conformation of the peptide inside the tunnel and leads to the shift of the A76 ribose. Further, 23S nucleotides 2504-2506 and 2452 adopt different conformations depending on the presence of erythromycin. These nucleotides form a part of the A site cavity and are shifted away from the P site in the presence of erythromycin. In the stalled complex, the A-site tRNA carries a charged lysine which in the simulations, interacts with these nucleotides and follows their movement. This movement in turn, increases the distance between the attacking alpha-amino group of the lysine and the carbonyl-carbon of the peptide by more than 1 A, suggesting a lower rate of peptide bond formation. Upon mutation of the lysine to an alanine, we observe a decrease of this distance. This decrease is in agreement with the observation that the mutation alleviates stalling. On the basis of the results, we propose that a mutation of the lysine to an arginine, would be expected to preserve stalling.

Mechanisms of ribosome stalling by SecM at multiple elongation steps.

Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway. Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling. However, the underlying molecular mechanism is still not clear at the atomic level. Here, we present two cryo-EM structures of the SecM-stalled ribosomes at 3.3-3.7 Å resolution, which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation. These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous, dynamic reshaping of the functional center of the ribosome.

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A

The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

The general mode of translation inhibition by macrolide antibiotics.

Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. To understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5'-proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The problematic sequence motifs are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segment that contacts the antibiotic molecule in the exit tunnel. Therefore, it appears that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not functioning during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics.

Drug Sensing by the Ribosome Induces Translational Arrest via Active Site Perturbation

During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site, leading to inhibition of peptide bond formation and translation arrest.

The Dynamics of SecM-Induced Translational Stalling

SecM is an E.coli secretion monitor capable ofstalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.

Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide

In bacteria, ribosome stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here we present a cryo-electron microscopy structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase centre (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural basis for understanding how the drug and the nascent peptide cooperate to inhibit peptide bond formation and induce translation arrest.

Crystallization of Escherichia coli 70S Ribosome in complex with tRNAs

The prokaryotic ribosome is responsible for translation, the process of creating polypeptides from amino acids. Specifically, ribosomes translate the codons in mRNAs into polypeptide chains. The prokaryotic ribosome is made up of a large subunit and a small subunit, which are known as the 50S (Svedberg units) and 30S subunits, respectively. The larger subunit catalyzes peptide bond formation within the peptidyl transferase center, while the smaller subunit mediates decoding of the mRNA. Together they make up the 70S prokaryotic ribosome. Studies have shown that certain nascent polypeptides can bind to the exit tunnel, resulting in inhibition of peptide bond formation which is referred to as ribosome stalling. Through the use of X‐Ray crystallography we will crystallize the 70S ribosome in complex with tRNAs and a nascent chain within the exit tunnel producing a structure. Although, no structure has been produced yet, we have been able to narrow down conditions that will optimize the growth of these crystals. This optimization will allow for production of the structure which will result in us being able to further explore the process of nascent chain mediated‐translational arrest in the Escherichia coli (E. coli) 70S ribosome.This research was supported in part by a grant from the Howard Hughes Medical Institute.

The Antibiotic Thermorubin Inhibits Protein Synthesis by Binding to Inter-Subunit Bridge B2a of the Ribosome

Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location—the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin's mode of action but possess improved pharmacodynamic properties.

Determination of the optimal position of adjacent proton-donor centers for the activation or inhibition of peptide bond formation – A computational model study

The study reports a computational analysis of the influence of proton donor group adjacent to the reaction center during ester ammonolysis of an acylated diol as a model reaction for peptide bond formation. This analysis was performed using catalytic maps constructed after a detailed scanning of the available space around the reaction centers in different transition states, a water molecule acting as a typical proton donor. The calculations suggest that an adjacent proton donor center can reduce the activation barrier of the rate determining transition states by up to 7.2 kcal/mol, while no inhibition of the reaction can be achieved by such a group.

Insights into the Chloramphenicol Inhibition Effect on Peptidyl Transferase Activity, Using Two New Analogs of the Drug

Chloramphenicol (CAM) inhibits peptide bond formation by binding to the 50S subunit of prokaryotic ribosomes and interfering competitively with the binding of the aminoacyl-tRNA 3'- terminus to ribosomal A-site. Further studies have demonstrated that CAM (I) reacts rapidly with a model initiator ribosomal complex (poly(U)-programmed ribosomes from Escherichia coli, bearing AcPhe-tRNA at the P-site), complex C, to form an encounter complex CI which is then isomerized slowly to a tighter complex, C  I. Herein, we show by time-resolved footprinting analysis that CAM produces a footprint in CI complex, comprising nucleotides A2451, G2505, and U2506, all exhibiting reduced reactivity against base-specific modifying agents. When C  I complex is footprinted, the reactivities of G2505 and U2506 are almost restored, while protection is observed at A2062 and altered reactivity at A2058 and A2059. Our results suggest that CAM initially binds to a hydrophobic crevice composed of nucleotides located adjacently to the A-site (CI complex). Soon after, CAM shifts slowly to a final position, in which the interaction between the p-nitrobenzyl group of CAM and the base of A2451 is conserved, while the dichloroacetyl group reorientates toward A2062. Analogous behavior is observed, if CAM is modified by replacement of dichloroacetyl group with � -alanyl. However, insertion at this position of a bulkier group, such as phenylalanyl-phenylalanyl group, sterically prevents CAM accommodation to its initial binding site and favors its direct fitting into the final binding pocket. Our data correlate well with recent crystallographic results regarding CAM binding on Thermus thermophilus and E. coli ribosomes.