Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1620-1642 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1727-1753.

1620

A Unique Mechanism of Glycine-Specific Inhibition

of Bacterial Translation by Bottromycin A

Inna A. Volynkina

1,2,a

*, Aleksandr A. Grachev

, Alexei Livenskyi

2,4,5

Daria K. Yagoda

, Pavel S. Kasatsky

, Olga A. Tolicheva

Ekaterina S. Komarova

1,6

, Alexey E. Tupikin

, Vera A. Alferova

6,8

Anastasiia O. Karakchieva

, Arina A. Nikandrova

2,6,9

, Mikhail V. Biryukov

Yuliya V. Zakalyukina

, Lubov V. Dorofeeva

, Yuriy A. Ikhalaynen

Igor A. Rodin

, Dmitrii A. Lukianov

1,2

, Marsel R. Kabilov

, Alena Paleskava

3,12

Andrey L. Konevega

3,12,13,b

*, Petr V. Sergiev

1,2,6,c

*, and Olga A. Dontsova

1,2,6,8

Department of Chemistry, Lomonosov Moscow State University, 119234 Moscow, Russia

Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology,

121205 Moscow, Russia

Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P. Konstantinov

of National Research Center “Kurchatov Institute”, 188300 Gatchina, Leningrad Region, Russia

Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119234 Moscow, Russia

A.N. Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119234 Moscow, Russia

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences,

630090 Novosibirsk, Novosibirsk Oblast, Russia

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

Department of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

Department of Soil Science, Lomonosov Moscow State University, 119234 Moscow, Russia

All-Russian Collection of Microorganisms (VKM), G. K. Skryabin Institute of Biochemistry and Physiology

of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences,

142290 Pushchino, Moscow Oblast, Russia

Institute of Biomedical Systems and Biotechnologies, St. Petersburg Peter the Great Polytechnic University,

195251 Saint Petersburg, Russia

Centre for Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Center),

National Research Center “Kurchatov Institute”, 123182 Moscow, Russia

e-mail: vialabgroup@gmail.com

e-mail: konevega_al@pnpi.nrcki.ru

e-mail: petya@genebee.msu.su

Received October 18, 2025

Revised November 6, 2025

Accepted November 6, 2025

Abstract— The rise of antimicrobial resistance among pathogenic bacteria poses a critical challenge to modern

medicine, highlighting an urgent need for novel therapeutic agents. Bottromycin A

(BotA2) is a promising

candidate for future drug development, demonstrating potent activity against clinically relevant pathogens,

including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Mycoplasma

species, although its molecular mechanism of action has remained unclear until now. Here, we demonstrate

that BotA2 inhibits bacterial translation with unique context specificity determined by the mRNA coding se-

quence. Using high-throughput toe-printing coupled with deep sequencing (Toe-seq analysis), we show that

BotA2 induces ribosome pausing predominantly when a glycine codon enters the A-site of the ribosome,

* To whom correspondence should be addressed.

BOTTROMYCIN A

MECHANISM OF ACTION 1621

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

regardless of the codon identities in the P- and E-sites. Our biochemical and biophysical data indicate that

BotA2 specifically arrests glycine-delivering ternary complexes on the ribosome, thereby preventing full ac-

commodation of incoming Gly-tRNA

Gly

within the peptidyl transferase center. Altogether, our findings uncover

a previously undescribed mechanism of translation inhibition, driven by the context-specific immobilization

of ternary complexes on elongating ribosomes.

DOI: 10.1134/S0006297925603740

Keywords: bottromycin, antibiotics, translation, inhibition of protein synthesis, context specificity, ternary

complex

INTRODUCTION

Bottromycin A

(BotA2) is a ribosomally synthe-

sised and post-translationally modified peptide, first

reported in 1957 [1]. It was purified from the fermen-

tation broth of Streptomyces bottropensis and found

to be active against Gram-positive bacteria [2] and

Mycoplasma species [3, 4]. Later on, other structural-

ly related compounds, named as bottromycin B

[2],

[5], and D [6] were discovered. Together with BotA2,

they constitute a unique class of macrocyclic peptide

antibiotics ‒ bottromycins ‒ gaining a special interest

nowadays as promising antimicrobial agents [7].

BotA2 demonstrates activity against a wide range

of bacterial strains [5]. Of particular interest is the ac-

tivity of BotA2 on methicillin-resistant Staphylococcus

aureus and vancomycin-resistant Enterococcus [8, 9],

as well as its moderate activity against Mycobacteri-

umsp. and Staphylococcus aureus clinical isolates re-

sistant to erythromycin, carbomycin, tetracycline, and

penicillin [2, 5]. Moreover, BotA2 has proven its effi-

ciency at combating mycoplasma infections in  vitro [3,

4, 10], being even more potent than classical therapeu-

tic agents, such as macrolides and tetracyclines, used

for the treatment of mycoplasmosis [11]. In addition,

BotA2 was also found to be active in vitro against the

Gram-negative phytopathogenic bacterium Xanthomo-

nas oryzae pv. oryzae, the causative agent of bacterial

leaf blight of rice [12]. This emphasizes the possibility

of BotA2 application in plant protection.

BotA2 is synthesized from the precursor peptide,

BtmD, through a series of post-translational modifi-

cations required to ensure antibacterial activity and

resistance to proteases [13]. However, the chemical

structure of mature BotA2 still has an Achilles’ heel,

which is the labile methyl ester (marked in red in

Fig.1a) readily hydrolyzed under physiological condi-

tions, e.g., in blood plasma, yielding a less active de-

methylated form of BotA2 [14]. Instability of BotA2 in

oral and parenteral administration [4, 15] constitutes

the main concern for introducing BotA2 into clinical

practice. At the same time, antibacterial properties of

BotA2 render it a promising scaffold in drug design

and prompt researchers to develop more stable BotA2

derivatives [14-22]. For instance, bottromycin A

hy-

drazide is much more stable in the bloodstream and

active invivo against Mycoplasma gallisepticum in the

infected chickens [4]. However, it demonstrated 8-16

times lower antibacterial activity in  vitro compared

to BotA2 [14]. Over time, the accumulated knowledge

allowed characterizing structure-activity relationships

of BotA2 and its derivatives, revealing that the ester

and thiazole moieties could be altered, while the rest

of the molecule is essential for antibacterial proper-

ties [21]. To date, only two synthesized BotA2 deriv-

atives ‒ propyl and ethyl ketones replacing the ester

group ‒ were shown to have both improved plasma

stability and antibacterial activity comparable to that

of the parental BotA2 [14]. These two compounds,

especially the propyl ketone, might be considered as

prospective candidates for future drug development.

While the biosynthesis and antibacterial activities

of BotA2 are well studied, its molecular mechanism

of action remains obscure. Previous works suggest

that BotA2 inhibits translation by targeting the bac-

terial ribosome [23,  24]. However, there have been

conflicting reports as to the ability of BotA2 to in-

hibit the puromycin reaction [25-28], and many pro-

posals that BotA2 exerts its influence either through

inhibiting EF-G-dependent translocation [26,  29] or

through lowering the affinity of aminoacyl-tRNAs

(aa-tRNAs) for the A-site [28, 30, 31]. In the earliest

works, the authors noted that the activity of BotA2

highly depends on the presence of C and G nucleo-

tides in the mRNA template [23, 25]. BotA2 was more

efficient at inhibiting in  vitro translation on poly(C),

poly(UC), and poly(UG) mRNAs, compared to poly(A)

and poly(U). Moreover, BotA2 was shown not to af-

fect aminoacylation of some tRNAs (tRNA

Leu

, tRNA

Phe

and tRNA

Pro

) [23]. Non-enzymatic binding of aa-tRNAs

(Pro-tRNA

Pro

, Phe-tRNA

Phe

) to ribosomes was also un-

influenced by BotA2 [23, 27, 32]. At the same time,

BotA2 was able to interfere with the binding of some

ternary complexes (aa-tRNA∙EF-Tu∙GTP) in a concen-

tration-dependent manner, with the inhibition rate be-

ing 30-50% [30, 31]. Furthermore, BotA2 was demon-

strated not to inhibit the puromycin-mediated release

of nascent peptides in the in vivo system based on

VOLYNKINA et al.1622

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Bacillus megaterium protoplasts [33]. Likewise, no

substantial inhibition of the puromycin reaction was

observed by using polysomes purified from Escherichia

coli cells, even when the reactions were supplement-

ed with elongation factor EF-G and GTP [31, 34]. Along

with this observation, BotA2-induced inhibition of

polypeptide synthesis was insensitive to the presence

of EF-G∙GTP, suggesting that BotA2 is unlikely to di-

rectly affect translocation [27,  30]. Not surprisingly,

BotA2 was shown not to affect EF-G-dependent GTP

hydrolysis [27]. Another important finding was that

the presence of an excess of 50S over 30S ribosomal

subunit decreased the inhibitory effect of BotA2 on

polypeptide synthesis, while such an effect was not

observed using an excess of 30S over 50S [24]. This

indicates that BotA2 presumably interacts with the

50S ribosomal subunit. However, all previous studies

do not allow one to draw an unambiguous conclusion

about the detailed mechanism of translation inhibi-

tion by BotA2.

Herein we present insights into the molecular

mechanism of action of BotA2. Using biochemical

and biophysical approaches, we uncovered that BotA2

inhibits bacterial translation demonstrating unique

context specificity determined by the mRNA coding

sequence. BotA2 was found to induce ribosome stall-

ing exclusively when a glycine codon is in the A-site

of the ribosome, with stalling efficiency independent

of the codon identities in the P- or E-sites. Delving

into the details of BotA2 action, we identified that

BotA2 does not target glycyl-tRNA synthetase and does

not provoke mistranslation of glycine codons, rath-

er completely abolishes peptide bond formation with

the incoming glycine residue. Furthermore, we show

that BotA2 does not interfere with EF-Tu-dependent

delivery of Gly-tRNA

Gly

to the ribosome, but instead

traps the ternary complex, preventing Gly-tRNA

Gly

from its full accommodation into the ribosomal A-site.

Taking into account amino acid specificity of BotA2,

our findings establish a novel, previously undescribed

mechanism of translation inhibition based on specific

immobilization of Gly-tRNA

Gly

in the unaccommodat-

ed (likely A/T) state on the ribosome.

MATERIALS AND METHODS

For more details on experimental procedures,

please refer to the Supplementary Methods in the

Online Resource 1.

Cultivation of the producing strain, fraction-

ation, and purification of BotA2 and BotCA. The

producing strain Streptomyces sp. VKM Ac-2945

was

provided from the All-Russian Collection of Microor-

ganisms, Pushchino, Russia. It was originally isolated

from soil collected in the Belgorod Region in 1993.

The strain was cultivated in 750  mL Erlenmeyer

flasks containing 200-250 mL of soy-glycerol medi-

um [2% soy flour, 1.5% glycerol, 0.2% yeast extract,

0.5% CaCl

, 0.1% NaCl, 0.1% K

HPO

, tap water, pH

7.2] at 28°C with constant shaking (200 rpm, Inno-

44 Shaker, New Brunswick Scientific, USA) until

the appearance of pronounced antibacterial activi-

ty (~10-12 days). The activity of fermentation broth

and eluted fractions was assessed using the E.  coli

lptD

mut

pDualrep2.1 reporter strain [35], as previ-

ously described [36]. The culture broth was further

separated from biomass by centrifugation at 20,000g

for 5 min and subjected to solid-phase extraction on

LPS-500-H sorbent with 120 μm particle size (Techno-

sorbent LLC, Russia) using water-acetonitrile mixtures

as eluents. Active fractions, containing BotCA (elut-

ed with 30-50% acetonitrile) and BotA2 (eluted with

75-100% acetonitrile) were collected and subjected

to preparative RP-HPLC.

Preparative RP-HPLC was performed on a

puriFlash

PF-4250 preparative chromatograph (In-

terchim, France) equipped with a VDSpher 100 C18-E

(10  µm, 20×250 mm) column (VDS optilab, Germany).

Chromatograms for BotA2- and BotCA-enriched frac-

tions are provided in Fig. S1 in the Online Resource1.

The identity of the isolated compounds was first con-

firmed by analytical RP-HPLC using BotA2 and BotCA

standards, and then by high-resolution mass spec-

troscopy (HRMS). HPLC-HRMS/MS analysis was per-

formed using the Orbitrap Exploris 240 mass spec-

trometer coupled with the Vanquish UHPLC system

(Thermo Fisher Scientific, USA), equipped with a re-

versed-phase Acclaim™ 120 C18 (2.2  µm, 2.1×150  mm)

column (Dionex, USA). For BotCA, the observed ions

were [M–H]

−

at m/z 807.4224 and [M+H]

at m/z

809.4366 (calculated for C

S: [M–H]

−

807.4227,

Δ  0.4  ppm; [M+H]

809.4384, Δ  2  ppm). For BotA2, the

observed ions were [M–H]

−

at m/z 821.4382 and [M+H]

at m/z 823.4528 (calculated for C

S: [M–H]

−

821.4384, Δ  0.2  ppm; [M+H]

823.4540, Δ  1.5  ppm).

The MS

fragmentation patterns of both compounds

are provided in Fig. S2 in the Online Resource 1 and

display all the characteristic fragment ions, consistent

with previous reports [37].

At the stage of preliminary experiments low-

resolution mass spectra were recorded using the

EXPEC L-Chrom MS triple quadrupole system (EXPEC

Technology, China) equipped with an electrospray

ionization source (ESI).

Agar diffusion assays. Agar diffusion assay on

a panel of E.  coli resistant mutants was carried out

according to the reported method [36]. Agar diffusion

assay using misreading error reporter systems was

performed as described previously [38].

Determination of minimum inhibitory concen-

tration (MIC). MIC values were determined using

BOTTROMYCIN A

MECHANISM OF ACTION 1623

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

the standard liquid broth microdilution assay [39]

in 96-well sterile plates and a total volume of 100μL

per well. Minimum inhibitory concentration was de-

fined as the lowest concentration of an examined

compound at which the growth of the bacterial

strain was completely inhibited. For each MIC mea-

surement, at least two biological replicates were per-

formed.

In vitro translation in bacterial cell-free sys-

tems. Translation reactions (3 μL total volume) were

carried out with the PURExpress

In vitro Protein

Synthesis Kit (New England BioLabs, USA) according

to the manufacturer’s protocol and previously report-

ed procedure [40]. Alternatively, the reactions (5 μL

total volume) were carried out with E.coli S30 Extract

System for Linear Templates (Promega, USA) accord-

ing to the manufacturer’s instructions and previously

reported assay [41].

In vitro translation in a mammalian cell-free

system. The whole cell extract was prepared from the

HEK293T cell line as described previously [42] with

minor modifications: harvested cells were not treat-

ed with lysolecithin buffer. Translation reactions were

carried out as reported previously [43, 44].

Toe-printing assay. Linear DNA templates RST1

and RST3 (Table S2 in the Online Resource  1) were

generated by PCR using the following pairs of par-

tially complementary primers – RST1-fwd  +  RST1-rev

and RST3-fwd  +  RST3-rev, respectively (Table  S1 in

the Online Resource  1). DNA templates containing

short ORFs (Table S2 in the Online Resource  1) were

generated by PCR using the pRFPCER plasmid [45]

as a template, the CER-R reverse primer (Table S1

in the Online Resource 1), and a set of different for-

ward primers, listed in the Table S1 in the Online

Resource  1. MG template was generated using the

M-GGC forward primer and NEW-CER-R reverse prim-

er. Radiolabeled NV1 and NV2 primers (Table S1 in

the Online Resource  1) were used to generate cDNA

fragments by reverse transcription. The toe-printing

analysis of drug-dependent ribosome stalling was per-

formed essentially as previously described [46] with

some minor modifications indicated in the Supple-

mentary Methods in the Online Resource 1.

RelE-printing assay. The RelE-printing analysis

was performed in a similar way as described for the

toe-printing assay, but with some modifications in-

dicated below. The purified RelE protein (0.8  mg/mL

stock solution) was kindly provided by Dr.  Dmitry  E.

Andreev, A.  N.  Belozersky Institute of Physico-Chem-

ical Biology, Lomonosov Moscow State University,

Moscow, Russia [47]. RelE was diluted 1  :  10 in Pure

System Buffer [9mM Mg(OAc)

, 5mM KH

, 95mM

potassium glutamate, 5  mM  NH

Cl, 0.5  mM  CaCl

1  mM spermidine, 8  mM  1,4-diaminobutane, 1  mM

DTT, pH  7.3] prior to the experiment. For control re-

actions, nuclease-free water was diluted 1  :  10 in Pure

System Buffer. Following the incubation of translation

reactions at 37°C for 15 min, 0.5 μL of the RelE solu-

tion or the control solution was added to each sample

and the incubation was continued at 37°C for 15 min.

Next, 1pmol of the [

P]-labeled NV2 primer (Table S1

in the Online Resource  1) and 2  U of the AMV Reverse

Transcriptase (Roche, Switzerland) were added, and

reaction tubes were additionally incubated at 37°C

for 15 min. The subsequent experimental procedure

was exactly the same as described in the Supple-

mentary Methods in the Online Resource 1 for the

toe-printing assay.

Toe-seq assay. The Toe-seq analysis of BotA2

context specificity was performed according to the

reported method [48]. Brief description of the exper-

imental procedure and computational processing of

Toe-seq data is provided in the Supplementary Meth-

ods in the Online Resource 1. Prepared NGS librar-

ies were sequenced on the MGIseq-2000 (MGI Tech,

China) at the Genomics Core Facility (ICBFM SB RAS,

Novosibirsk, Russia). Parameters of the resulting Toe-

seq datasets are given in the Table S3 in the Online

Resource 1.

Metabolic labeling assay. Overnight culture of

E.  coli lptD

mut

was diluted to an OD

600

of 0.1 in MOPS

minimal medium containing 0.4% glycerol without

antibiotics and grown at 37°C for 1  h. Then, 50  μL of

[

P]-orthophosphoric acid (1  mCi/mL, 8500  Ci/mmol)

was added to 1  mL of E.  coli culture to achieve a fi-

nal radioactivity of 50  μCi/mL, followed by incubation

at 37°C for 50  min. Bottromicyn  A

, microcin  C, and

mupirocin were added to E.  coli cells and incubation

continued for 30  min at 37°C. Final concentrations of

antibiotics were 20  μM for bottromycin  A

and mi-

crocin  C, and 60  μM for mupirocin, which exceed the

corresponding MICs. Metabolically radiolabeled nucle-

otides were extracted with formic acid and subjected

to thin-layer chromatography as previously reported

[49], for subsequent detection by autoradiography.

Global analysis of tRNA aminoacylation level

(GATRAL). Overnight culture of E. coli lptD

mut

was

diluted 1 : 50 in fresh Lysogeny Broth (LB) medium

without antibiotics and grown at 37°C until an OD

600

of 0.4 was reached. Bottromycin A

, microcin C, or no

additive (control) was added to E. coli cells, followed

by an additional incubation at 37°C for 30 min. For

both antibiotics, the final concentration was 20 μM,

which exceeds the corresponding MICs. Then, tRNA

fractions were isolated from 5 mL of culture using

the Total RNA and Small RNA Isolation Kit (Biolabmix,

Russia), according to the manufacturer’s protocol.

Aminoacyl-tRNAs were acetylated with acetic anhy-

dride, hydrolyzed via RNase digestion, and analyzed

by LC-QTOF-MS, according to the published proce-

dure [50].

VOLYNKINA et al.1624

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Sample preparation for self-assembled in  vitro

translation. In  vitro translation system on the basis

of individually purified components was assembled

in the buffer TAKM

[50  mM Tris-HCl (pH  7.5), 70  mM

Cl, 30  mM  KCl, 7  mM  MgCl

] essentially as previ-

ously described [51] with some modifications indi-

cated below. MG (AUG-GGC), MF (AUG-UUU), and MV

(AUG-GUU) mRNAs were obtained by T7 transcription

followed by purification on a HiTrap

  Q  HP anion ex-

change column (Cytiva, USA) [52]. DNA templates for

in  vitro transcription were amplified by PCR using

the following pairs of primers ‒ T7-fwd-1  +  NV2 (for

MG) and T7-fwd-2  +  LP-rev (for MF and MV). DNA

sequences of primers and templates are provided in

Tables S1 and S2 in the Online Resource  1, respec-

tively. Individual tRNA

Gly

was prepared as previously

reported [53] with some modifications provided in

the Supplementary Methods in the Online Resource1.

Fluorescently labeled tRNA

Gly

(Prf16/17/20) was pre-

pared exactly as described previously [54]. Amino-

acylated fMet-tRNA

fMet

, BODIPY-Met-tRNA

fMet

, Phe-

tRNA

Phe

, and Val-tRNA

Val

were purified by RP-HPLC

and stored at −80°C. Gly-tRNA

Gly

(Prf16/17/20) was

prepared immediately before ternary complex for-

mation. For this, 10  µM tRNA

Gly

(Prf16/17/20) was

incubated with 0.2  mM  Gly, 3  mM  ATP, 2  mM  DTT,

0.75  µM  Gly-tRNA synthetase in the TAKM

buffer at

37°C for 40 min. Where indicated, ternary complex-

es were additionally incubated with bottromycin A

or kirromycin at 37°C for 5 min prior to the experi-

ment. Final concentrations of antibiotics after mixing

initiation complex with ternary complex were 100μM

and 150 μM for bottromycin A

and kirromycin, re-

spectively.

In  vitro synthesis of fluorescently labeled pep-

tides. In  vitro synthesis of short fluorescently labeled

peptides was performed essentially as recently de-

scribed [52, 55] with some modifications indicated be-

low. Linear DNA templates (0.3  pmol) were expressed

in a cell-free bacterial transcription-translation cou-

pled system using the PURExpress

Δ(aa, tRNA) Kit

(New England BioLabs) in a total volume of 5 μL,

according to the manufacturer’s guidance. Final con-

centrations of reagents in translation mixtures were

corrected for the sake of better representation as in-

dicated below. Each reaction consisted of 0.8 μL of

Solution A (minus aa, tRNA, from the kit), 1.2 μL of

Solution B (from the kit), 0.53 μM E.  coli Ribosomes

(New England BioLabs), 0.16μM BODIPY-Met-tRNA

fMet

0.2  μL of uncharged tRNA (7  mg/mL), 0.3  mM  L-ser-

ine (Sigma-Aldrich, USA), 0.3  mM  L-glycine (Sigma-

Aldrich), 2  U of RiboLock RNase Inhibitor (Thermo

Fisher Scientific), 15  ng of a DNA template, and

50  μM bottromycin A

. Negative control samples (in-

dicated as “−”) were supplemented with nuclease-free

water instead of bottromycin A

. In parallel, control

reactions were assembled to synthesize the follow-

ing fluorescently labeled peptides – BODIPY-Met,

BODIPY-Met-Ser, BODIPY-Met-Ser-Gly, and BODIPY-

Met-Ser-Gly-Phe. Control reactions consisted of 1 μL

of Solution A (minus aa, tRNA, from the kit), 1.3 μL

of Solution B (from the kit), 0.08 μM BODIPY-Met-

tRNA

fMet

, 0.35  μL of uncharged tRNA (7  mg/mL),

0.3  mM L-serine (Sigma-Aldrich), 0.3  mM L-glycine

(Sigma-Aldrich), 0.3  mM L-phenylalanine (Sigma-

Aldrich), 2  U of RiboLock RNase Inhibitor (Thermo

Fisher Scientific), and 10  ng of a DNA template

(M-AGT, M-AGT-GGC, or M-AGT-GGC-F, TableS2 in the

Online Resource  1). The “BODIPY-Met” sample was

supplemented with nuclease-free water instead of a

DNA template. Before the addition of templates, all

reaction tubes were pre-incubated at room tempera-

ture (RT) for 5min and then placed back on ice. Fol-

lowing the addition of templates, reaction mixtures

were incubated at 37°C for 30  min.

For the synthesis of short fluorescently labeled

peptides using the self-assembled in  vitro transla-

tion system, initiation complexes were programmed

with either of the three mRNAs ‒ MG (AUG-GGC), MF

(AUG-UUU), and MV (AUG-GUU) ‒ and assembled with

BODIPY-Met-tRNA

fMet

in the P-site. Ternary complex-

es were assembled with the following tRNAs ‒ Gly-

tRNA

Gly

, Phe-tRNA

Phe

, and Val-tRNA

Val

. Translation

was initiated by adding 0.2  µM ternary complex to

0.1  µM initiation complex followed by incubation

at 37°C for 2 min.

Translation reactions were terminated by the ad-

dition of 0.29  M NaHCO

to hydrolyze peptidyl-tRNA

and release synthesized peptides, followed by incu-

bation at 37°C for 20  min. Then, the samples were

mixed with an equal volume of Formamide Loading

Dye [98% formamide, 10mM EDTA (pH  8.0), 0.1% bro-

mophenol blue], incubated for 3 min at 70°C, and re-

solved in a 20×20 cm 12% PAAG containing 7  M urea

in TBE buffer [90  mM Tris base, 90  mM boric acid,

2  mM EDTA, pH 8.3]. The gel was scanned using the

Typhoon™ FLA 9500 Biomolecular Imager (GE Health-

care, USA). Laser of 473  nm (blue LD laser) was used

for excitation and DBR1 filter of 530  ±  20  nm was

used for emission. Images were processed and visu-

alized using the Image  Lab™ software (version 6.0.1,

Bio-Rad Laboratories, USA).

Rapid kinetics measurements. To monitor

the time course of aminoacyl-tRNA interaction with

the A-site of the ribosome, we rapidly mixed either

(i)  0.1  µM initiation ribosome complexes containing

BODIPY-Met-tRNA

fMet

programmed with the MG mRNA

with 1  µM Gly-tRNA

Gly

∙EF-Tu∙GTP ternary complex at

20°C or (ii)  0.1  µM Gly-tRNA

Gly

(Prf16/17/20)∙EF-Tu∙GTP

ternary complex with 0.4  µM initiation ribo-

some complexes programmed with the same MG

mRNA at 20°C. In both cases, samples were mixed

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in equal volumes (60  µL). Fluorescence was record-

ed using SX-20 stopped-flow spectrometer (Applied

Photophysics, UK). Proflavin fluorescence was excit-

ed at 460  nm, BODIPY FL fluorescence was excited

at 470  nm. Fluorescence intensity was measured after

passing a cut-off filter KV 495 nm (Schott, Germany)

in both cases. Time courses were obtained by aver-

aging 5-7 individual traces. Data were evaluated by

fitting to a single-exponential function with a char-

acteristic apparent rate constant (k

app

), amplitude(A),

and final signal amplitude (F

∞

) according to equation

F  =  F

∞

  +  A  ∙  exp(k

app

  ∙  t), where F is the fluorescence at

time t. Where necessary, two exponential terms were

used. All calculations were performed using the Graph-

Pad Prism software (version 9.3.1, Dotmatics, USA).

Monitoring of EF-Tu coelution with the ri-

bosomes. Reaction mixture containing 1 µM initi-

ation complexes programmed with the MG mRNA

and 2 µM Gly-tRNA

Gly

(Prf16/17/20)∙EF-Tu∙GTP ternary

complex was incubated at 37°C for 2  min followed

by separation on size exclusion chromatography col-

umn (BioSuite 450  Å HR SEC (8 μm, 7.8  ×  300 mm),

Waters, USA) in TAKM

buffer. 1 pmol of the central

fraction of 70S peak was resolved by SDS-PAGE, and

the presence of EF-Tu was assessed by western blot-

ting using primary monoclonal anti-His

antibodies

(His-Tag Antibody, Affinity Biosciences, China) and sec-

ondary horseradish peroxidase–conjugated antibodies

(Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP,

Thermo Fisher Scientific). The bands were developed

using the Clarity™ Western ECL Substrate (Bio-Rad

Laboratories) and visualized using the chemilumines-

cence mode in the ChemiDoc™ MP Imaging System

(Bio-Rad Laboratories).

RESULTS

Bottromycin  A

selectively inhibits bacterial

translation. Bottromycin  A

(BotA2, Fig. 1a) has long

Fig. 1. Bottromycin A

inhibits bacterial translation in a context-specific manner. a) Chemical structures of bottromycinA

(BotA2) and its hydrolyzed counterpart, referred to here as bottromycin A

carboxylic acid (BotCA). b) BotA2 and BotCA

inhibit bacterial protein synthesis in vitro. The efficiency of translation reaction (relative maximum Fluc accumulation

rates) is plotted vs. antibiotic concentration. Error bars represent standard deviation, n≥2. The calculated IC

values and

95% confidence intervals are shown in the table. c) Toe-printing analysis of BotA2 and BotCA on RST1 and RST3 mRNAs.

Sequences of the corresponding ORFs and the encoded amino acids are shown on the right. Asterisk (*) in the translated

sequence denotes a stop codon. Blue and orange diamonds mark the toe-printing bands corresponding to ribosomes stalled

during translation. Codons occupying the A-site of the stalled ribosomes are highlighted in the same color. Thiostrepton

(Ths) was included to map the translation start site. Borrelidin (Borr) arrests ribosomes at Thr codons. Atibiotics were

added to the final concentration of 50μM.

VOLYNKINA et al.1626

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

been known to inhibit protein synthesis in bacterial

cells [23], however, the details of its action remained

obscure. Since clinical application of BotA2 is limited

due to the poor stability under physiological condi-

tions, probably caused by hydrolysis of the methyl es-

ter group [14], we were interested in comparing the

action of BotA2 and its demethylated counterpart –

bottromycin  A

carboxylic acid (BotCA, Fig. 1a) –

in a cell-free bacterial in  vitro translation system.

By using the commercially available PURExpress

system reconstituted from purified Escherichia coli

translation components, we observed a dose-depen-

dent inhibition of protein synthesis by both BotA2

and BotCA, with the latter one being two-fold less

efficient (IC

  =  6.4  ±  0.8  μM and 13.4  ±  2.0  μM, re-

spectively, Fig.  1b). At the same time, BotCA was

previously reported to exhibit 64 times less potency

against Gram-positive strains compared to BotA2 [14],

suggesting that the main reason for the low in  vivo

activity of BotCA may be its impaired ability to pen-

etrate bacterial cells rather than inability to suppress

translation.

By using an alternative cell-free in  vitro system

based on the E.  coli S30 extract, we observed a strong

translation inhibition by BotA2 with IC

  =  1.0  ±  0.1  μM

(Fig.  S3 in the Online Resource  1), which is compa-

rable to other potent translation inhibitors, such as

tetracenomycin  X  [56], chloramphenicol [57], eryth-

romycin [58], linezolid [59], and madumycin  II  [60]

that display IC

values of 1.5  μM, 2.1  μM, 0.32  μM,

1.1  μM, and 0.3  μM, respectively. In addition, we have

tested whether BotA2 and BotCA can interfere with

eukaryotic mRNA translation. Both of them showed

no inhibition of protein synthesis in the HEK293T

whole cell lysate even at 50  μM concentrations, with

BotA2 exhibiting only limited inhibitory effect at high-

er concentrations (500-1000  μM) (Fig.  S4 in the Online

Resource  1). These observations provide evidence that

bottromycin  A

specifically targets the machinery of

bacterial translation.

Bottromycin  A

does not exhibit cross-resis-

tance to most known 50S-targeting antibiotics.

Since BotA2 was shown to inhibit protein synthesis,

we decided to test whether its binding site overlaps

with that of other known ribosome-targeting antibi-

otics. By using an agar diffusion assay, we screened

our homemade collection of resistant mutants for the

growth inhibition induced by BotA2. Surprisingly, we

have not found any E.  coli mutant resistant to BotA2

(Fig.  S5 in the Online Resource  1), which is consistent

with previous works showing no cross-resistance be-

tween BotA2 and erythromycin, as well as tetracycline

[5, 61]. Our data reveal that the BotA2 action is in-

sensitive to the following nucleotide substitutions in

the 23S rRNA ‒ U1782C, G2057A, A2058G, A2059G,

A2062G, U2586G, U2586C, U2609G, while they confer

strong resistance to erythromycin, chloramphenicol,

or tetracenomycin X (Fig.S5 in the Online Resource1).

In addition, we checked whether the Cfr-medi-

ated methylation of A2503 in the 23S rRNA would

confer resistance to BotA2, as it has been shown for

chloramphenicol, lincosamides, oxazolidinones, pleu-

romutilins, and streptogramins  A  [62]. Antibacterial

activity of BotA2 was measured against the Bacil-

lus subtilis 168 strain engineered to express the cfr

gene [63], in comparison with the susceptible strain

(Table  S4 in the Online Resource  1). As expected, cfr

expression resulted in an 8-fold increase in the mini-

mum inhibitory concentration (MIC) of chloramphen-

icol (MIC  =  100  μM). At the same time, we observed

no significant differences in MIC values for BotA2 be-

tween the two B.  subtilis strains. This result implies

that BotA2 does not interact with the A2503 residue.

Our reasoning is additionally supported by one of the

previous works, which revealed that BotA2 does not

compete with chloramphenicol for the binding site

[25]. Altogether, these findings suggest that BotA2,

being a potent translation inhibitor, does not share

binding site with classical 50S-targeting antibiotics,

but instead occupies a unique binding site.

Bottromycin A

inhibits translation in a con-

text-specific manner. Since the previous investi-

gation reported that BotA2 inhibits aminoacyl-tRNA

(aa-tRNA) binding in the A-site of the ribosome [31],

we decided to visualize which step of translation is

inhibited by BotA2 using the toe-printing assay. Ap-

plication of RST1 and RST3 mRNA templates revealed

that BotA2 induces ribosome stalling during the elon-

gation step, and specifically when a glycine (Gly) co-

don in the mRNA reaches the A-site of the ribosome

(Fig. 1c). This observation contradicts the expectations

for a conventional inhibitor of aa-tRNA accommoda-

tion, such as tetracycline, kirromycin, or lincosamides

(lincomycin, clindamycin) [64]. They generate multiple

toe-printing bands at the beginning of mRNA coding

sequence accompanied by pronounced ribosomal ar-

rest at the start codon  [65]. Translation reactions on

RST3 mRNA supplemented with borrelidine (Borr), an

inhibitor of threonyl-tRNA synthetase, revealed that a

fraction of ribosomes bypasses the BotA2 stalling site

and becomes trapped downstream, at the Thr codon

occupying the A-site (Fig.  1c). We assume that BotA2

may bind to its target reversibly, as previously pro-

posed [25].

The obtained results prompt us to comprehen-

sively examine the BotA2 context specificity. A recent-

ly developed high-throughput technique named Toe-

seq was applied for this purpose [48]. Toe-seq uses

a library of short DNA templates containing a 30-nt

randomized region within its ORF. In  vitro expression

of this library using a coupled transcription-trans-

lation system in the presence of BotA2, followed

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Fig.  2. Toe-seq analysis of bottromycin A

context specificity. a)  pLogo analysis of BotA2-induced ribosome stalling sites.

Amino acids corresponding to codons positioned in the E-, P-, and A-sites of arrested ribosomes are indicated. The n(fg)

and n(bg) values represent the number of foreground and background sequences used to generate the image, respectively.

As the foreground, we used stalling sites identified in BotA2-treated samples. As the background, we used 100,328 possible

3-aa motifs extracted from the same datasets. b) pLogo analysis of a subset of BotA2-induced ribosome stalling sites, in

which glycine (Gly, G) is fixed as the A-site amino acid (position +1). aandb)Red horizontal lines on the pLogo correspond

to p = 0.05. c)  Enrichment of codons occupying the A-site of the ribosomes stalled in the presence of BotA2. Enrichment

is shown as the mean of normalized relative occurrence of codons among 12,463 identified stalling sites in two biological

replicates. Negative values conform to underrepresented codons, while positive values conform to overrepresented ones.

Only codon positions 4-11 of the ORF are included in the analysis, as they correspond to the variable region of mRNA.

Error bars indicate standard deviation.

by treatment with AMV reverse transcriptase re-

sulted in multiple parallel toe-printing experiments.

The generated cDNA fragments were subsequently

subjected to next-generation sequencing (NGS) to

identify ribosome stalling sites and map them to the

mRNA library. Toe-seq analysis was performed with

50  μM BotA2 in two biological replicates along with

untreated control reactions. Datasets obtained for an-

tibiotic-treated samples were normalized to the con-

trols and filtered according to predefined criteria (see

Supplementary Methods in the Online Resource  1 for

details). The resulting MaxStallProbability scores, rep-

resenting the relative efficiency of antibiotic-induced

ribosome stalling, were well correlated between both

replicates (Fig.  S6 in the Online Resource  1). To de-

termine whether specific sequence signatures were

associated with the sites of BotA2-mediated transla-

tion stalling, we applied pLogo analysis [66] to 12,245

sites identified in two biological replicates. Analysis of

amino acid residues associated with codons occupying

the E-, P-, and A-sites of arrested ribosomes revealed

a strong bias toward Gly as the incoming amino acid

(Fig. 2a), consistent with our earlier toe-printing ex-

periments (Fig. 1c). Additionally, a marginal, but de-

tectable enrichment was observed for alanine (Ala)

codons positioned in the A-site. At the same time, ri-

bosome stalling showed no clear dependence on the

last or penultimate amino acid residue of the nascent

polypeptide chain. Fixing Gly or Ala at the position

+1 of the pLogo plot revealed some minor preferences

for amino acid residues at positions −1 and 0 (Fig.  2b,

Fig.  S7 in the Online Resource  1). However, the signif-

icance of these preferences remains questionable and

requires further validation.

We were also interested to analyze whether the

efficiency of BotA2-induced ribosome stalling depends

on individual codons more than on encoded amino

acids. To this end, we calculated the relative occur-

rence of individual codons among the identified stall

sites and noticed that different A-site Gly codons ex-

hibit different stalling frequencies, with GGC being

the most efficient one, followed by GGG (Fig.  2c).

VOLYNKINA et al.1628

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Fig. 3. Bottromycin A

-induced ribosome stalling depends only on the identity of the A-site codon and is unrelated to

inhibition of tRNA

Gly

aminoacylation. BotA2-mediated specificity of ribosomal arrest does not depend on the identity of

the P-site codon (a), but depends on the identity of the A-site Gly codon (b). a and b)  Toe-printing analysis of BotA2 on

two sets of short mRNAs containing variable codons. Sequences of the corresponding ORFs and the encoded amino acids

are shown on the right. Asterisk (*) denotes a stop codon. Blue diamonds mark the toe-printing bands corresponding to

BotA2-induced ribosome stalling. Thiostrepton (Ths) was included to map the translation start site. Atibiotics were added

to the final concentration of 50 μM. c)  Addition of BotA2 does not change the amount of different aa-tRNAs in bacterial

cells. Fold change in the level of aminoacyl-adenosines derived from aa-tRNAs that were purified from E. coli lptD

mut

cells

treated with bottromycinA

(BotA2) or microcinC (McC) is shown. The values obtained for antibiotic-treated samples were

normalized to the values obtained from untreated cells. Error bars represent standard deviation, n = 3.

Similarly, among the A-site Ala codons, GCC was as-

sociated with more frequent translation arrest com-

pared to GCU, GCA, and GCG. At the same time, there

was no statistically significant enrichment of codons

occupying the E- and P-sites of the stalled ribosomes.

These data, along with the distribution of ribosome

stalling sites across the mRNA library, are provided in

the Online Resource2. Altogether, the Toe-seq analysis

confirmed that BotA2 acts as a context-specific inhib-

itor of bacterial translation, arresting ribosomes spe-

cifically when a Gly codon occupies the A-site.

To further validate the sequence specificity of

BotA2 observed in Toe-seq data, we set up a con-

ventional toe-printing system using a set of model

mRNAs. Thus, short DNA templates encoding fMet-

Xxx-Gly peptides (where Xxx denotes a variable ami-

no acid), were prepared to assess the contribution of

the P-site codon to BotA2-mediated ribosome stalling.

Since the GGC codon was associated with the majority

of ribosome stalls, we selected it to encode Gly. Co-

dons corresponding to the 2

amino acid (Xxx) were

randomly selected so that some of them were asso-

ciated with high MaxStallProbability scores, while

others – with medium and low scores (Fig.  S8 in the

Online Resource  1). Toe-printing analysis of the gener-

ated DNA templates in the presence of BotA2 revealed

ribosome stalling at the 2

position, regardless of the

P-site codon identity (Fig.  3a). The absence of any re-

liable difference in BotA2-induced translation arrest

on these templates is in agreement with our earlier

Toe-seq results (Fig. 2).

Next, we checked whether the efficiency of ri-

bosome stalling induced by BotA2 depends on the

identity of Gly codons, as predicted by Toe-seq analy-

sis (Fig. 2c). Two sets of short DNA templates were

prepared to be translated into fMet-Gly-Phe and

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fMet-Ser-Gly-Phe peptides, where Gly amino acid is

encoded by different nucleotide triplets – GGC, GGG,

GGA, or GGU. Both sets of templates showed that GGC

and GGG codons were almost equally efficient, while

GGU exhibited the least pronounced ribosomal arrest

(Fig.  3b, Fig.  S9 in the Online Resource  1). Interest-

ingly, GGA demonstrated a strong toe-printing band,

being placed at the 2nd position in ORF (Fig.  S9 in

the Online Resource  1), while at the 3rd position, it

was much less efficient at stalling ribosomes (Fig.  3b).

This observation might be related to previous results

showing that GGA considerably decreases translation

efficiency in E.  coli cells, when it is placed right after

the start codon, and does not alter translation being

placed at the downstream positions [67, 68]. Probably,

other factors unrelated to BotA2 action enhance ribo-

some stalling at the start codon, when Gly-tRNA

Gly

used as a substrate [67, 69].

Bottromycin A

does not inhibit glycyl-tRNA

synthetase. One possible explanation of a unique

sequence specificity of BotA2 action could be the

inhibition of tRNA

Gly

aminoacylation by the antibi-

otic. To test this hypothesis, we used two different

approaches. Typical inhibitors of aminoacyl-tRNA

synthetases, such as protein kinase HipA [70], mi-

crocin  C  [71], mupirocin  [72], and serine hydroxam-

ate  [73], induce RelA-dependent synthesis of gua-

nosine tetraphosphate (ppGpp). In cells, amino acid

starvation leads to a shortage of free ternary com-

plexes along with the accumulation of deacylated

tRNAs. The RelA protein specifically recognizes an

uncharged tRNA in the A-site of a stalled ribosome,

acting as a sensor of amino acid deficiency [74, 75].

Upon activation, RelA converts cellular ATP and GTP/

GDP to the alarmone ppGpp [76], thus inducing the

stringent response aimed at the upregulation of genes

involved in amino acid biosynthesis, protein hydroly-

sis, and transition to a so-called “hibernation” state as

a means of energy conservation and survival [77-79].

By using the metabolic labeling of BotA2-sus-

ceptible E.  coli lptD

mut

strain, we examined whether

BotA2 will induce the accumulation of ppGpp. Bacte-

rial cells were grown in the medium supplemented

with [

P]-radiolabeled orthophosphoric acid, followed

by treatment with BotA2. As reference agents, we

used microcin  C (McC) and mupirocin (Mup), inhib-

itors of aspartyl-tRNA [80] and isoleucyl-tRNA syn-

thetases [81], respectively. After 30 min incubation,

the labeled nucleotides were extracted with formic

acid and separated via thin-layer chromatography

(TLC). While McC and Mup provoked the accumula-

tion of ppGpp in cells, BotA2 had no detectable effect

(Fig.  S10 in the Online Resource  1). This observation

suggests that BotA2 does not increase the concentra-

tion of deacylated tRNAs and hence does not induce

the stringent response.

To directly assess whether BotA2 affects the in-

tracellular pool of aa-tRNAs, global analysis of tRNA

aminoacylation level (GATRAL) [50] was performed on

E.  coli lptD

mut

treated with BotA2. Microcin  C (McC)

was used as a reference agent. Total tRNA was isolat-

ed after 30 min incubation with antibiotics, followed

by N-acetylation of aa-tRNAs to stabilize amino acid

residues bound to the 3’-terminal adenosines. Then,

the samples were digested with a mixture of RNase I

and RNase  T1 to obtain N-acetylated aminoacyl-ade-

nosines (Ac-aa-Ade) that can be quantitatively ana-

lysed by LC-MS. The calculated fold change in the

amount of each Ac-aa-Ade, relative to the untreated

cells, reflects the abundance of the corresponding

aa-tRNA in the cells (Fig.3c). As expected, McC caused

a substantial decrease in the Asp-tRNA

Asp

level (see

Asp in Fig.  3c), while BotA2 had no effect, even on

Gly-tRNA

Gly

(see Gly in Fig.  3c). This analysis confirms

that BotA2 does not affect aminoacylation of tRNA

Gly

as well as other tRNAs.

In principle, BotA2 could theoretically target ami-

noacylated tRNAs directly, analogous to GNAT-family

toxins that specifically modify the aminoacyl moiety

[82]. However, a direct and specific interaction be-

tween this small antibiotic molecule and the acceptor

stem of aa-tRNA seems mechanistically implausible.

In addition, any non-N-acetylating modifications of

amino acid residues of aa-tRNA would likely disturb

the levels of acetylated glycyl-adenosines (Ac-Gly-Ade)

used in the GATRAL assays, while the structural fea-

tures of BotA2 (see Fig.  1a) rule out its function as an

acetylating agent. Taken together, these observations

strongly suggest that BotA2 neither inhibits glycyl-

tRNA synthetase, nor directly modifies Gly-tRNA

Gly

Bottromycin  A

does not induce mistranslation

of glycine codons. Another possible mechanism could

involve BotA2 specifically preventing Gly-tRNA

Gly

from binding to the A-site of the ribosome, increas-

ing its availability for erroneous binding of near-cog-

nate aa-tRNAs. If this were the case, BotA2 would be

expected to provoke translational misreading of Gly

codons similarly to other error-inducing antibiotics

[83]. To assess the ability of BotA2 to decrease the

fidelity of translation, we employed a set of report-

er constructs encoding β-galactosidase variants with

different substitutions at the catalytic residue Glu537.

Under normal translation, reporter cells produce a

defective protein uncapable of catalyzing the hydro-

lysis of the X-Gal substrate. However, treatment with

certain antibiotics (e.g., kanamycin or streptomycin),

which increase the translational error rate, allows Glu

to be incorporated at the 537th position, resulting in

the synthesis of active β-galactosidase, detected by an

insoluble, indigo blue colored product [84]. Using an

agar diffusion assay, we tested BotA2, BotCA, and ref-

erence antibiotics on these reporter strains (Fig.  S11

VOLYNKINA et al.1630

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

in the Online Resource 1). As expected, we observed

blue halos upon treatment with kanamycin and strep-

tomycin, while rifampicin, an inhibitor of bacterial

RNA polymerase  [85], showed no coloration. Likewise,

BotA2 and BotCA failed to produce blue halos, even

when tested on pJC27-GGA and pJC27-GGG reporters,

in which the 537

GAA codon was replaced by GGA

or GGG, respectively. This observation led us to con-

clude that BotA2 does not provoke misreading of Gly

codons.

Bottromycin  A

does not interfere with the de-

livery of Gly-tRNA

Gly

to the A-site of the ribosome.

To determine a particular step of translation inhibit-

ed by BotA2, we examined whether it can specifical-

ly interfere with the delivery of Gly-tRNA

Gly

to the

A-site of the ribosome. For this purpose, we employed

a modified toe-printing assay, in which translation re-

actions were additionally treated with the RelE tox-

in, known to cleave mRNA in the ribosomal A-site

(Fig. 4a) [86]. Since the hydrolytic activity of RelE re-

quires a vacant A-site, RelE-printing serves as a useful

tool to monitor the occupancy of the A-site during

translation  [47]. Two mRNAs, whose translation is

highly susceptible to BotA2-induced stalling, were

selected for RelE-printing analysis of BotA2 action.

As expected, we observed the RelE-prints originating

from initiating and terminating ribosomes (see cleav-

age at the 2

and 5

codons, Fig.  4b), since initia-

tion complexes, pre-termination and post-termination

complexes possess unoccupied A-sites [87]. Notably,

the addition of thiostrepton (Ths) yielded only one

RelE-print corresponding to ribosomes trapped at the

start codon, consistent with the antibiotic’s mode of

action [88, 89]. Crucially, no RelE-prints corresponding

to mRNA cleavage at Gly codons were detected. These

data suggest that BotA2 allows delivery of Gly-tRNA

Gly

to the A-site of the ribosome, thereby preventing the

RelE-mediated cleavage of mRNA at Gly codons.

Bottromycin A

inhibits peptidyl transfer to

Gly-tRNA

Gly

. Having determined that the delivery of

Gly-tRNA

Gly

was not suppressed by BotA2, we sought

to uncover which of the following steps ‒ Gly-tRNA

Gly

accommodation, peptide bond formation, or translo-

cation ‒ is affected by BotA2. Some of the previous

works reported impaired translocation [26, 29], while

others stated that the inhibition of a peptide bond for-

mation may be the primary action of BotA2 [27, 28].

To discriminate between these two hypotheses, we de-

cided to monitor peptide formation in the presence

of BotA2 by employing a recently developed method

based on in  vitro synthesis of fluorescently labeled

peptides [52, 55].

Short mRNAs from the previous experiments

were used to synthesise BODIPY-labeled peptides in

the presence of BotA2 (Fig.  4c). To reduce the effect

of multiple-round translation, we decided to “freeze”

translating ribosomes at the Gly codon by excluding

Phe ‒ the last amino acid in the tetrapeptide – from

the reaction. To our surprise, the full-length BODIPY-

Met-Ser-Gly-Phe tetrapeptide was synthesized anyway,

probably due to the traces of Phe in the PURExpress  Δ

(aa, tRNA) Kit. Some amino acids or aminoacyl-ade-

nylates (aa-AMP) may co-purify with aminoacyl-tRNA

synthetases [90] during the kit preparation, thereby

compromising our experiments with short mRNA

templates. Nevertheless, after all the contaminating

Phe had been depleted, we were able to observe the

formation of truncated peptides originating from ar-

rested ribosomes. In case BotA2 had inhibited trans-

location, we would have expected to see similar

BODIPY-Met-Ser-Gly tripeptide yields regardless of the

BotA2 addition. Instead, we observed the inhibition

of BODIPY-Met-Ser-Gly synthesis in all reactions sup-

plemented with BotA2 (Fig.  4c). In agreement with

our earlier results (Fig.  3b), GGG and GGC codons

were associated with the highest inhibitory effect,

while GGU was the least efficient. The accumulation

of the BODIPY-Met-Ser dipeptide along with the inhi-

bition of BODIPY-Met-Ser-Gly synthesis indicates that

BotA2 primarily inhibits peptide bond formation rath-

er than translocation.

However, due to the traces of Phe in the PUREx-

press in  vitro translation system, the real inhibition

rate of peptide transfer to Gly-tRNA

Gly

was difficult to

assess. We decided to validate our observations using

a reconstituted in  vitro translation system consisting

of individual purified components [52]. The reaction

mixtures were assembled by combining initiating 70S

ribosomes attached to the mRNA with the AUG start

codon in the P-site followed by the codon directing

incorporation of Gly (GGC), Phe (UUU), or Val (GUU),

and the cognate ternary complex (aa-tRNA∙EF-Tu∙GTP),

containing Gly-tRNA

Gly

, Phe-tRNA

Phe

, or Val-tRNA

Val

respectively. Consistent with the previous experiment,

BotA2 prevented the formation of BODIPY-Met-Gly

entirely, while it had no effect on the synthesis of

BODIPY-Met-Phe and BODIPY-Met-Val (Fig. 4d). Alto-

gether, our findings strongly suggest that BotA2 specif-

ically impairs incorporation of glycine into a growing

peptide chain.

Bottromycin  A

prevents accommodation of

Gly- tRNA

Gly

in the peptidyl transferase center.

Since peptidyl transfer to Gly-tRNA

Gly

positioned in

the A-site is impaired in the presence of BotA2, we

considered whether BotA2 inhibits peptide bond for-

mation directly or instead prevents the accommoda-

tion of Gly-tRNA

Gly

in the peptidyl transferase cen-

ter (PTC) prior to transpeptidation. To verify these

possibilities, we tracked the approaching of accep-

tor stem of Gly-tRNA

Gly

to the PTC. To this end, we

used a stopped-flow fluorescence detection assay for

monitoring the delivery of glycine in the form of the

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Fig. 4. Bottromycin A

allows delivery of Gly-tRNA

Gly

to the A-site of the ribosome, but interferes with the peptide bond

formation. a)Schematic representation of RelE-mediated cleavage of mRNA. RelE binds in the ribosomal A-site and cleaves

mRNA after the 2nd or 3rd nucleotide in the A-site codon [86]. RelE does not cleave free mRNA, as well as when the A-site

is occupied with aa-tRNA. b)RelE-printing analysis of BotA2-induced ribosome stalling. Toe-printing reactions supplemented

with the RelE toxin indicate the position of ribosomes with the vacant A-site. Sequences of the corresponding ORFs and the

encoded amino acids are shown on the right. Asterisk(*) denotes a stop codon. Blue diamond marks the toe-printing bands

corresponding to BotA2-induced ribosome stalling. Green and red diamonds mark the RelE-printing bands corresponding to

mRNA fragments cleaved at the 2nd and 5th codons, respectively. Thiostrepton (Ths) was included to map the translation

start site. Antibiotics were added to the final concentration of 50μM. c andd) In vitro synthesis of fluorescently labeled pep-

tides using PURExpress(c) or self-assembled(d) translation system. In(c), each reaction was supplied with BODIPY- labeled

fMet-tRNA

fMet

, uncharged tRNAs, and amino acids ‒ Ser and Gly. BotA2 was added to the final concentration of 50 μM.

In (d), BODIPY-Met-Gly/Phe/Val dipeptides were formed upon addition of a pre-assembled cognate ternary complex to the

70S initiation complexes programmed with the MG (AUG-GGC), MF (AUG-UUU), or MV (AUG-GUU) mRNA and containing

BODIPY-Met-tRNA

fMet

in the P-site. Where indicated, ternary complexes were additionally incubated with 100 μM BotA2.

VOLYNKINA et al.1632

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig. 5. Bottromycin A

prevents accommodation of Gly-tRNA

Gly

in the peptidyl transferase center (PTC) by trapping ternary

complex on the ribosome. a and b) Stopped-flow experiments showing that BotA2 prevents accommodation of Gly-tRNA

Gly

in the ribosomal A-site. a) Pre-steady state kinetics of Gly-tRNA

Gly

accommodation monitored by fluorescence change of

BODIPY-Met-tRNA

fMet

positioned in the P-site of the 70S initiation complex in the absence (blue) or presence of BotA2

(red). b) Pre-steady state kinetics of Gly-tRNA

Gly

binding to the A-site monitored by fluorescence change of the ternary

complex Gly-tRNA

Gly

(Prf16/17/20)·EF-Tu·GTP upon interaction with the 70S initiation complex. Time courses were obtained

in the absence (blue) or presence of BotA2 (red), as well as in the presence of kirromycin (Kirr, green) or modified variant

of EF-Tu (His84Ala) deficient on GTP hydrolysis (purple). c and d) BotA2 impairs EF-Tu dissociation from the translating

ribosome. c) Profiles of size exclusion chromatography of the 70S initiation complex after incubation with the ternary

complex Gly-tRNA

Gly

(Prf16/17/20)∙EF-Tu∙GTP in the presence of BotA2. Absorbance values were measured at 260 nm (blue).

Fluorescence was measured using excitation at 460 nm and detection at 510 nm (red). TC, ternary complexes. d) Upper

panel: SDS-PAGE of His-tagged EF-Tu (lane EF-Tu) and 70S peak fractions corresponding to ribosomal complexes assembled

in the absence (lane ‒) or presence of BotA2 (lane BotA2). Lower panel: Immunoblotting of the area between the dashed

lines with anti-His

antibodies. Where indicated, BotA2 was added to the final concentration of 100μM.

ternary complex Gly-tRNA

Gly

∙EF-Tu∙GTP in the vicini-

ty of BODIPY-labeled methionine residue of initiator

tRNA positioned in the P-site of the ribosome (Fig.5a).

Interestingly, for BotA2-treated complexes we did not

observe the conventional increase in fluorescent sig-

nal, characteristic for the A-site accommodation of

aa-tRNA acceptor end [51]. This observation allowed

us to suppose that BotA2 does not inhibit peptidyl

transferase reaction per  se, but instead impairs the

preceding step of amino acid delivery to the PTC.

To monitor the delivery of glycine residue in

real time, we continued using the stopped-flow tech-

nique and prepared a modified tRNA

Gly

, labeled

with the fluorescent dye at the elbow region ‒

tRNA

Gly

(Prf16/17/20). The interaction of proflavin-la-

beled tRNA with the ribosomal A-site is well charac-

terized and can be divided into several substeps [91].

An increase in a typical biphasic fluorescence kinetic

curve cumulatively reflects the initial binding of the

ternary complex to the ribosome, followed by codon

recognition, which induces GTPase activation and re-

sults in GTP hydrolysis by EF-Tu. The subsequent de-

crease in fluorescence signal is related to the release

of aa-tRNA from the GDP-bound form of EF-Tu and

accommodation of the aa-tRNA in the ribosomal PTC

(Fig. 5b). The exponential fitting of the fluorescence

BOTTROMYCIN A

MECHANISM OF ACTION 1633

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

kinetic curves provided apparent rate constants of

the reactions preceding GTP hydrolysis and aa-tRNA

accommodation. Remarkably, BotA2 slightly reduced

the rate of ternary complex binding (k

app1

  (no  drug)  =

12.6  ±  0.7  s

−1

vs. k

app1

(BotA2)  =  7.9  ±  0.2  s

−1

), and com-

pletely abolished the following step – accommodation

of Gly-tRNA

Gly

Bottromycin  A

arrests glycine-delivering ter-

nary complexes on the ribosome. Taken together,

our findings imply two major possibilities. The first

one is that BotA2 hinders productive tRNA accom-

modation by forming a steric obstacle or changing

the conformation of the ribosome that closes the ac-

commodation corridor for tRNA after successful de-

coding and dissociation of EF-Tu. However, it is hard

to imagine that the effect can be pronounced in the

case of the smallest amino acid, glycine, whereas

bulky amino acids, such as phenylalanine or valine,

effectively pass through and participate in the pepti-

dyl transferase reaction. The second scenario involves

retention of the aa-tRNA in a bent conformation (A/T

state) with anticodon bound to mRNA and the accep-

tor stem interacting with EF-Tu, leading to the arrest

of the ternary complex on the ribosome. To discrim-

inate between these two possibilities, we performed

size exclusion chromatography of arrested ribosom-

al complexes, which allowed us to separate all the

components according to their hydrodynamic radii.

As expected, 70S ribosomes eluted first showing the

largest peak visualized by absorbance at 260  nm (blue

profile, Fig.  5c). Proflavin-labeled Gly-tRNA

Gly

, detect-

ed by fluorescence, was present both in the ribosomal

fraction and in the central part of the chromatogram

corresponding to ternary complexes (TC), proteins,

and tRNAs (red profile, Fig.  5c). Size exclusion chro-

matography profiles of untreated ribosomal complex-

es are provided in Fig.  S12 in the Online Resource  1.

To check whether EF-Tu was trapped on the ribosome

upon addition of BotA2, we analyzed the ribosomal

fraction by SDS-PAGE and immunoblotting with an-

ti-His

antibodies against His-tagged EF-Tu (Fig.  5d).

Clear EF-Tu band appeared only in the case of antibi-

otic-treated ribosomes, suggesting that BotA2 impairs

EF-Tu dissociation from the translating ribosome.

DISCUSSION

In this study, we examined the molecular mecha-

nism of the peptide antibiotic bottromycin  A

(BotA2),

a potent inhibitor of bacterial translation, whose

mode of action has long remained obscure despite

numerous reports. Here, we showed that BotA2 has

unique context specificity determined by the mRNA

coding sequence. We revealed that BotA2 stalls elon-

gating ribosomes predominantly at sites where a Gly

codon occupies the ribosomal A-site, with efficiency

independent of the codon identities in the P- and

E-sites. Looking for the explanations of this unique

context specificity, we proved that BotA2 neither in-

terferes with aminoacylation of tRNAs

Gly

nor provokes

the misreading of Gly codons. At the same time, the

peptide bond formation between the incoming Gly-

tRNA

Gly

and the peptidyl group of the P-site-bound

peptidyl-tRNA was completely abolished in the pres-

ence of BotA2. Furthermore, we demonstrated that

BotA2 traps Gly-delivering ternary complexes on the

ribosome, with the tRNA anticodon base-paired to

the mRNA in the A-site and unaccommodated accep-

tor stem of Gly-tRNA

Gly

likely retained in complex

with EF-Tu.

In addition, we showed that the binding site

ofBotA2 differs from those of other known ribosome-

targeting antibiotics. Although previous study suggest-

ed that BotA2 binds to the 50S ribosomal subunit [24],

it does not exhibit cross-resistance to erythromycin,

chloramphenicol, tetracenomycin X, lincosamides, and

other antibiotics targeting peptidyl transferase center

(Fig.  S5, Table  S4 in the Online Resource  1). Selection

of resistant mutants is considered to be the gold stan-

dard for identifying the binding site of an antibiotic.

We repeatedly attempted to select E. coli mutants re-

sistant to BotA2 using strains carrying either all seven

rRNA operons or a single copy of them – an approach

suitable for the selection of rRNA mutations. However,

these efforts consistently yielded either a bacterial

lawn or no viable clones (data not shown). While

these observations highlight the therapeutic potential

of BotA2, our findings suggest that BotA2 binds in the

unique site, distinct from those of classical 50S-target-

ing translation inhibitors.

From this, one may assume that BotA2 interacts

with the ternary complex, rather than with the ri-

bosome itself. Several antibiotics are known to bind

to elongation factors and trap them on the ribosome.

For example, kirromycin restricts EF-Tu structural

rearrangements and freezes the factor in its active

GTP-bound conformation, even after the GTP hydro-

lysis, preventing aa-tRNA accommodation [92, 93]. A

similar mechanism has been shown for other kir-

romycin-like antibiotics (e.g., aurodox, efrotomycin,

factumycin) [94,  95], enacyloxin IIa [96], as well as

for didemnin B and ternatin-4 targeting the eukary-

otic homolog of EF-Tu – eEF1A [97, 98]. Importantly,

these drugs arrest ternary complexes regardless of

the tRNA identity, consistent with their binding sites

at the interface of domain  I (also known as G-domain)

and domain  III of EF-Tu or eEF1A [99-102]. The speci-

ficity of BotA2 for one particular amino acid could be

reasoned by a novel and previously unreported mech-

anism emanating from interactions between the anti-

biotic and the acceptor stem of Gly-tRNA

Gly

positioned

VOLYNKINA et al.1634

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig. 6. The proposed mechanism of bottromycin A

-induced ribosome stalling. Schematic diagram illustrating that bottro-

mycin A

(BotA2) selectively traps glycine-delivering ternary complexes on the ribosome, preventing the accommodation

of Gly-tRNA

Gly

in the peptidyl transferase center.

in the amino acid binding pocket of EF-Tu. One could

imagine that BotA2 associates only with EF-Tu, deliv-

ering the smallest amino acid, while larger incoming

residues would sterically clash with the antibiotic.

Being associated, BotA2 probably “glues” Gly-tRNA

Gly

to EF-Tu, thereby impairing EF-Tu dissociation from

the ribosome and preventing Gly-tRNA

Gly

accommoda-

tion in the PTC (Fig. 6).

A comprehensive understanding of how BotA2

interacts with its target requires in-depth structur-

al analysis complemented by genetic approaches of

target validation, making the involvement of multiple

research groups beneficial and essential. This study

was initiated five years ago as a collaborative effort

between our laboratory – including Dr.  Ilya  A.  Oster-

man, who later withdrew from the project – and Prof.

Rolf  Müller, Dr.  Joy  Birkelbach (both from the Helm-

holtz Institute for Pharmaceutical Research, Germa-

ny), and Prof. Daniel N. Wilson (University of Ham-

burg, Germany). We also gratefully acknowledge our

collegial interaction with Dr.  Yury  S.  Polikanov (Uni-

versity of Illinois, Chicago, USA), to whom we provid-

ed our sample of BotA2 in early 2022 along with our

preliminary findings. In 2025, Drs.  Yury  S.  Polikanov

and Dmitry  Y.  Travin (University of Illinois, Chicago,

USA) reciprocated by sharing their (then) unpublished

data on the structure of the BotA2-ribosome com-

plex and BotA2 resistance mutations, which provid-

ed compelling evidence for BotA2 direct interaction

with Gly-tRNA

Gly

bound to EF-Tu [103]. Their findings

complemented our own earlier results and motivated

us to extend the study with the last assay detecting

BotA2-induced trapping of Gly-containing ternary

complexes on translating ribosomes.

Building on our initial observation of BotA2 Gly-

specific activity, the combined structural, genetic, bio-

chemical, and biophysical data from both research

teams have now converged to establish a unique

mechanism of translation inhibition by BotA2, in-

volving the context-specific immobilization of terna-

ry complexes on elongating ribosomes. This article

summarizes our contributions toward clarifying the

molecular details of this mechanism.

In an effort to explain why BotA2 exhibits prefer-

ences for some Gly codons, we analyzed whether this

might be related to codon usage or the abundance

of corresponding tRNAs (Table  S5 in the Online Re-

source  1). In E.  coli, codons GGU and GGC are over-

represented in highly expressed genes, while GGA

and GGG are extremely underrepresented [104]. This

pattern correlates well with the intracellular concen-

tration of tRNA

Gly

species. Three isoaccepting tRNAs

Gly

are encoded in the E.  coli genome [105]: codons GGC

and GGU are recognized by tRNA

Gly

GCC

(hereafter

Gly3), GGA is recognized exclusively by tRNA

Gly

UCC

(Gly2), while GGG is decoded by both tRNA

Gly

UCC

and

tRNA

Gly

CCC

(Gly1) (Table S5 in the Online Resource 1).

Consistent with codon usage, Gly3 is the most abun-

dant tRNA

Gly

in E.  coli cells, followed by Gly2 and

Gly1. Probably, the evolutionary depletion of GGA and

GGG codons reflects their resemblance to the Shine–

Dalgarno sequence, which could impede productive

BOTTROMYCIN A

MECHANISM OF ACTION 1635

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

translation elongation [106]. However, all these data

do not correlate with the BotA2 codon preferences.

The most plausible explanation is that the

strength of the codon-anticodon interaction modu-

lates the efficiency of BotA2-induced ribosome stalling

(Fig. S13 in the Online Resource 1). Apparently, per-

fect codon-anticodon Watson-Crick pairing stabilizes

Gly-tRNA

Gly

within the arrested complex. For exam-

ple, Gly1 and Gly3 pair perfectly with GGC and GGG

codons, respectively, through the formation of a total

of 9 hydrogen bonds. In contrast, Gly2 forms only 8

hydrogen bonds when pairing with GGA or GGG co-

dons. However, the 5-methylaminomethyl modification

of uridine (mnm

U) at the first anticodon position of

Gly2 has been shown to stabilize pairing with both

A and G bases [84,  107]. Therefore, the GGA and

GGG recognition by Gly2 leads to the formation of

only slightly less stable codon-anticodon duplexes. As

such, the weakest interaction is anticipated with Gly3

pairing to GGU, which forms 8 hydrogen bonds, not

stabilized by anticodon modifications (Fig. S13 in the

Online Resource 1). To summarize, Gly codons can

be ranked as GGC  ≥  GGG  >  GGA  >  GGU, based on the

strength of codon-antibodon interactions. This hierar-

chy correlates well with BotA2 preferences and helps

to explain why GGU is the most abundant Gly codon

in highly expressed genes (~60%). It is conceivable

that a less stable base pairing increases the probabil-

ity of Gly-tRNA

Gly

dissociation from the ribosome as

a part of a complex with EF-Tu and BotA2. Inhibition

of aa-tRNA accommodation and peptide bond forma-

tion likely compels ternary complexes delivering less

“sticky” Gly-tRNAs

Gly

to undergo repeated rounds of

association/dissociation, thereby increasing the ribo-

some’s chances to bypass the stalling motif. A sim-

ilar mechanism has recently been shown for chlor-

amphenicol’s action [108]. Thus, the proposed model

connects the affinity of codon-anticodon base-pairing

with the efficiency of BotA2-induced ribosome stalling.

Interestingly, the btmD gene, which encodes the

bottromycin precursor peptide, begins with the evo-

lutionary conserved ATG-GGA-CCC-… codons, trans-

lated into the fMet-Gly-Pro-… amino acid sequence

[109]. Since BotA2 specifically stalls ribosomes at Gly

codons, we assume that it might limit its own bio-

synthesis via a negative feedback loop. As previously

predicted, the bottromycin(btm) gene cluster contains

only one self-resistance gene, btmA, which encodes a

major facilitator superfamily (MFS) transporter, prob-

ably responsible for BotA2 secretion. It is quite likely

that when the concentration of BotA2 in the fermen-

tation broth reaches a certain level, the intracellular

concentration of the antibiotic also starts to increase

until equilibrium. High levels of BotA2 inside the pro-

ducing cells may specifically lead to downregulation

of btmD translation, thereby inhibiting further BotA2

production to ensure cellular survival. In addition,

the BtmH epimerase has been shown to bind excess

BotA2 [110], presumably providing additional protec-

tion against self-poisoning [13]. On one hand, BtmH

can sequester excess BotA2 with high affinity, thus

lowering its free intracellular concentration. On the

other hand, bound BotA2 directly inhibits BtmH, pre-

venting further maturation of new BotA2 molecules

[110]. Together, these mechanisms might control BotA2

yield upon fermentation to avoid self-harm.

As previously reported, BotA2 is not stable under

physiological conditions and degrades in plasma more

rapidly than it reaches the focus of inflammation [15].

At the same time, a series of BotA2 derivatives have

been synthesized, some of which demonstrate both

increased stability and promising antibacterial activi-

ty [14]. We hope that our work will facilitate new ef-

forts to develop improved BotA2 derivatives, and new

BotA2-based medications will soon be available for

patients suffering from multidrug resistant pathogens.

Supplementary information

The online version contains supplementary materials

available at https://doi.org/10.1134/S0006297925603740.

Acknowledgments

We express our sincere gratitude to Prof.  Rolf Müller

and Dr. Joy Birkelbach at the Helmholtz Institute for

Pharmaceutical Research Saarland (HIPS) and Helm-

holtz Centre for Infection Research (HZI) (Saarbrück-

en, Germany) for providing standards for bottromy-

cin A

and bottromycin A

carboxylic acid. We thank

Prof.  Daniel  N.  Wilson (Institute for Biochemistry and

Molecular Biology, University of Hamburg, Hamburg,

Germany) for cooperation in early stages of the proj-

ect development and valuable discussions. We are

especially grateful to Dr.  Ilya  A.  Osterman, who orig-

inally conceived this project, for supporting early

aspects of this work. We also acknowledge the work

of Yury  S.  Polikanov (University of Illinois, Chicago,

IL, USA) and his colleagues, who have shared their

data ahead of publication. We also thank Andrey  G.

Tereshchenkov and Dr.  Natalia  V.  Sumbatyan at the

Lomonosov Moscow State University (Moscow, Rus-

sia) for helpful suggestions and attempts to reveal the

binding site of bottromycin A

. We thank Elizaveta  A.

Razumova for assistance with E.  coli ΔtolC pJC27 re-

porter cells. We thank Dr.  Dmitry  E. Andreev at the

A.  N.  Belozersky Institute of Physico-Chemical Biology

(Lomonosov Moscow State University, Moscow, Russia)

for providing the purified RelE protein and discuss-

ing results. We thank Tinashe  P. Maviza for provid-

ing E. coli SQ171 strains transformed with different

pLK35 and pAM552 plasmids. We thank the staff at

the Skoltech Advanced Mass Spectrometry Core Fa-

cility (Moscow, Russia) for performing LC-QTOF-MS

VOLYNKINA et al.1636

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

analysis, especially Maria Zavialova and Dmitriy

Maslov. Preparation of uncharged total tRNA, meta-

bolic labeling, and analysis of aminoacylation levels

of tRNAs were performed by Alexei Livenskyi, whose

work was supported by the grant of Russian Science

Foundation [24-14-00181]. HPLC-HRMS/MS analysis

was supported by Lomonosov Moscow State Univer-

sity Program of Development.

Contributions

I.A.V.: validation, formal analysis, data curation,

software, investigation (in vitro translation assays,

toe-printing, RelE-printing, misreading reporter assay,

synthesis of BODIPY-peptides with PURExpress sys-

tem), writing – original draft, visualization, project

administration. A.A.G.: formal analysis, investigation

(synthesis of BODIPY-peptides with self-assembled

in vitro translation system, rapid kinetics measure-

ments, monitoring of EF-Tu coelution with the ribo-

somes). A.L.: methodology, validation, formal analysis,

data curation, investigation (metabolic labeling assay,

GATRAL), writing – original draft, visualization. D.K.Y.:

investigation (in vitro translation assays, toe-printing,

RelE-printing, growth inhibition of resistant mutants,

synthesis of BODIPY-peptides with PURExpress system,

solid-phase extraction). P.S.K.: investigation (tRNA

Gly

purification, self-assembled in vitro translation sys-

tem). O.A.T.: investigation (synthesis of BODIPY-pep-

tides with self-assembled in vitro translation system).

E.S.K.: methodology, investigation (in  vitro translation

assays, Toe-seq), funding acquisition. A.E.T.: investiga-

tion (NGS). V.A.A.: methodology, validation, data cu-

ration, resources, investigation (RP-HPLC purification

of BotA2 and BotCA), writing – original draft, visual-

ization. A.O.K.: investigation (toe-printing, MIC mea-

surement, solid-phase extraction). A.A.N.: data cura-

tion, investigation (solid-phase extraction, analytical

RP-HPLC). M.V.B., Y.V.Z., and L.V.D.: methodology, in-

vestigation (cultivation of the producing strain), writ-

ing – review & editing. Y.A.I. and I.A.R.: validation,

investigation (HPLC-HRMS/MS). D.A.L.: methodology,

resources, writing – review & editing, project admin-

istration. M.R.K.: formal analysis, resources, data cu-

ration, software. A.P.: methodology, validation, formal

analysis, data curation, writing– original draft, visual-

ization, funding acquisition. A.L.K.: conceptualization,

resources, writing – review &  editing, supervision,

project administration. P.V.S.: conceptualization, for-

mal analysis, resources, writing – review &  editing,

supervision, project administration. O.A.D.: resources,

supervision, funding acquisition. All authors read and

approved the final manuscript.

Funding

This work was supported by Russian Science Founda-

tion (RSF), grant number 21-64-00006-P to O.A.D (bac-

terial in vitro translation, toe-printing, RelE-printing,

Toe-seq, experiments with bacterial cells), grant num-

ber 25-14-00253 to A.P. (experiments on self-assem-

bled translation system, pre-steady state kinetics, and

EF-Tu trapping), and grant number 24-74-00057 to

E.S.K. (eukaryotic in vitro translation). The funders

had no role in study design, data collection and analy-

sis, decision to publish, or manuscript preparation.

Data availability

The Toe-seq sequencing data have been deposited to

the NCBI BioProject database under accession number

PRJNA1304769. Bioinformatics scripts are available

at https://github.com/kabilov/Publication_scripts/tree/

main/2024_Toe-seq. Other relevant data that support

this study are available in the main manuscript, sup-

plementary information, or from the corresponding

authors upon reasonable request.

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

Open access

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any medium or format, as long as you give appro-

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you will need to obtain permission directly from the

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