Coli Ribosome Research Articles

A computational workflow to determine drug candidates alternative to aminoglycosides targeting the decoding center of E. coli ribosome

The global antibiotic resistance problem necessitates fast and effective approaches to finding novel inhibitors to treat bacterial infections. In this study, we propose a computational workflow to identify plausible high-affinity compounds from FDA-approved, investigational, and experimental libraries for the decoding center on the small subunit 30S of the E. coli ribosome. The workflow basically consists of two molecular docking calculations on the intact 30S, followed by molecular dynamics (MD) simulations coupled with MM-GBSA calculations on a truncated ribosome structure. The parameters used in the molecular docking suits, Glide and AutoDock Vina, as well as in the MD simulations with Desmond were carefully adjusted to obtain expected interactions for the ligand-rRNA complexes. A filtering procedure was followed, considering a fingerprint based on aminoglycoside's binding site on the 30S to obtain seven hit compounds either with different clinical usages or aminoglycoside derivatives under investigation, suggested for in vitro studies. The detailed workflow developed in this study promises an effective and fast approach for the estimation of binding free energies of large protein-RNA and ligand complexes.

Minimization of the E. coli ribosome, aided and optimized by community science.

The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.

Diaphorin, a polyketide produced by a bacterial endosymbiont of the Asian citrus psyllid, adversely affects the in vitro gene expression with ribosomes from Escherichia coli and Bacillus subtilis.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria), an obligate mutualist of an important agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera). Our previous study demonstrated that diaphorin, at physiological concentrations in D. citri, inhibits the growth and cell division of Bacillus subtilis (Firmicutes) but promotes the growth and metabolic activity of Escherichia coli (Gammaproteobacteria). This unique property of diaphorin may aid microbial mutualism in D. citri, potentially affecting the transmission of "Candidatus Liberibacter spp." (Alphaproteobacteria), the pathogens of the most destructive citrus disease Huanglongbing. Moreover, this property may be exploited to promote microbes' efficiency in producing industrial materials. However, the mechanism underlying this activity is unknown. Diaphorin belongs to the family of pederin-type compounds, which inhibit protein synthesis in eukaryotes by binding to eukaryotic ribosomes. Therefore, as a first step to assess diaphorin's direct influence on bacterial gene expression, this study examined the effect of diaphorin on the in vitro translation using ribosomes of B. subtilis and E. coli, quantifying the production of the green fluorescent protein. The results showed that the gene expression involving B. subtilis and E. coli ribosomes along with five millimolar diaphorin was 29.6% and 13.1%, respectively, less active than the control. This suggests that the diaphorin's adverse effects on B. subtilis are attributed to, at least partly, its inhibitory effects on gene expression. Moreover, as ingredients of the translation system were common other than ribosomes, the greater inhibitory effects observed with the B. subtilis ribosome imply that the ribosome is among the potential targets of diaphorin. On the other hand, the results also imply that diaphorin's positive effects on E. coli are due to targets other than the core machinery of transcription and translation. This study demonstrated for the first time that a pederin congener affects bacterial gene expression.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA.

Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for identifying 5-methylcytidine in DNA. This account focuses on the chemistry of bisulfite with pseudouridine, an isomer of the RNA nucleoside uridine in which the uracil base is connected to C1' of ribose via C5 instead of N1. Pseudouridine, Ψ, is the most common nucleotide modification found in cellular RNA overall, in part due to its abundance in rRNAs and tRNAs. It has a stabilizing influence on RNA structure because N1 is now available for additional hydrogen bonding and because the heterocycle is slightly better at π stacking. The isomerization of U to Ψ in RNA strands is catalyzed by 13 different enzymes in humans and 11 in E. coli; some of these enzymes are implicated in disease states which is testament to the biological importance of pseudouridine in cells. Recently, pseudouridine came into the limelight as the key modification that, after N1 methylation, enables mRNA vaccines to be delivered efficiently into human tissue with minimal generation of a deleterious immunogenic response. Here we describe the bisulfite reaction with pseudouridine which gives rise to a chemical sequencing method to map the modified base in the epitranscriptome. Unlike the reaction with cytidine, the addition of bisulfite to Ψ leads irreversibly to form an adduct that is bypassed during cDNA synthesis by reverse transcriptases yielding a characteristic deletion signature. Although there were hints to the structure of the bisulfite adduct(s) 30 to 50 years ago, it took modern spectroscopic and computational methods to solve the mystery. Raman spectroscopy along with extensive NMR, ECD, and computational work led to the assignment of the major product as the (R) diastereomer of an oxygen adduct at C1' of a ring-opened pseudouridine. Mechanistically, this arose from a succession of conjugate addition, E2 elimination, and a [2,3] sigmatropic rearrangement, all of which are stereodefined reactions. A minor reaction with excess bisulfite led to the (S) isomer of a S-adducted SO3- group. Understanding structure and mechanism aided the design of a Ψ-specific sequencing reaction and guided attempts to improve the utility and specificity of the method. Separately, we have been investigating the use of nanopore direct RNA sequencing, a single-molecule method that directly analyzes RNA strands isolated from cells after end-ligation of adaptor sequences. By combining the electrical current and base-calling data from the nanopore with dwell-time analysis from the helicase employed to deliver RNA to the nanopore, we were able to map Ψ sites in nearly all sequence contexts. This analysis was employed to find Ψ residues in the SARS-CoV-2 vRNA, to analyze the sequence context effects of mRNA vaccine synthesis via in vitro transcription, and to evaluate the impact of stress on chemical modifications in the E. coli ribosome. Most recently, we found that bisulfite treatment of RNA leading to Ψ adducts could modulate the nanopore signal to help in mapping modifications of low occupancy.

Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates

As genetic code expansion advances beyond l-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-l-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi–Dunitz angle of 76–115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.

Direct nanopore sequencing for the 17 RNA modification types in 36 locations in the E. coli ribosome enables monitoring of stress-dependent changes

Direct nanopore sequencing for the 17 RNA modification types in 36 locations in the E. coli ribosome enables monitoring of stress-dependent changes

MetaD minima for the paper "Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates"

MetaD minima for the paper "Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates"

Computational data for the paper "Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates"

Computational data for the paper "Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates"

RiboDraw: semiautomated two-dimensional drawing of RNA tertiary structure diagrams

Publishing, discussing, envisioning, modeling, designing and experimentally determining RNA three-dimensional (3D) structures involve preparation of two-dimensional (2D) drawings that depict critical functional features of the subject molecules, such as noncanonical base pairs and protein contacts. Here, we describe RiboDraw, new software for crafting these drawings. We illustrate the features of RiboDraw by applying it to several RNAs, including the Escherichia coli tRNA-Phe, the P4–P6 domain of Tetrahymena ribozyme, a −1 ribosomal frameshift stimulation element from beet western yellows virus and the 5′ untranslated region of SARS-CoV-2. We show secondary structure diagrams of the 23S and 16S subunits of the E. coli ribosome that reflect noncanonical base pairs, ribosomal proteins and structural motifs, and that convey the relative positions of these critical features in 3D space. This software is a MATLAB package freely available at https://github.com/DasLab/RiboDraw.

Cross-Linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics.

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas-phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) bovine serum albumin (BSA), (ii) Escherichia coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1623 unique cross-links for nuclear lysates, corresponding to 1088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving three-dimensional (3D) structures of single proteins but also for performing system-wide protein interaction studies.

Investigation of radezolid interaction with non-canonical chloramphenicol binding site by molecular dynamics simulations

Radezolid is a promising antibiotic of oxazolidinone family, which is able to overcome effect of some linezolid resistance mechanisms of bacterial ribosomes. The structure of the radezolid complex with ribosomes was never published but, by analogy with linezolid, it is considered to prevent the binding of aminoacyl–tRNA to the A–site of the ribosome large subunit. However, as with linezolid, it can be assumed that radezolid binds to the alternative binding site existing in the A,A/P,P–ribosome. In the present article we have investigated this issue by molecular dynamics simulations and proposed the structure of the radezolid complex with a E. coli ribosome, which is consistent with available data of biochemical investigations of radezolid action.

Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity

The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

Thermodynamic Cost, Speed, Fluctuations, and Error Reduction of Biological Copy Machines.

Due to large fluctuations in cellular environments, transfer of information in biological processes without regulation is error-prone. The mechanistic details of error-reducing mechanisms in biological copying processes have been a subject of active research; however, how error reduction of a process is balanced with its thermodynamic cost and dynamical properties remain largely unexplored. Here, we study the error reducing strategies in light of the recently discovered thermodynamic uncertainty relation (TUR) that sets a physical bound to the cost-precision trade-off for dissipative processes. We found that the two representative copying processes, DNA replication by the exonuclease-deficient T7 DNA polymerase and mRNA translation by the E. coli ribosome, reduce the error rates to biologically acceptable levels while also optimizing the processes close to the physical limit dictated by TUR.

Kinetic proofreading and the limits of thermodynamic uncertainty.

To mitigate errors induced by the cell's heterogeneous noisy environment, its main information channels and production networks utilize the kinetic proofreading (KPR) mechanism. Here, we examine two extensively studied KPR circuits, DNA replication by the T7 DNA polymerase and translation by the E. coli ribosome. Using experimental data, we analyze the performance of these two vital systems in light of the fundamental bounds set by the recently discovered thermodynamic uncertainty relation (TUR), which places an inherent trade-off between the precision of a desirable output and the amount of energy dissipation required. We show that the DNA polymerase operates close to the TUR lower bound, while the ribosome operates ∼5 times farther from this bound. This difference originates from the enhanced binding discrimination of the polymerase which allows it to operate effectively as a reduced reaction cycle prioritizing correct product formation. We show that approaching this limit also decouples the thermodynamic uncertainty factor from speed and error, thereby relaxing the accuracy-speed trade-off of the system. Altogether, our results show that operating near this reduced cycle limit not only minimizes thermodynamic uncertainty, but also results in global performance enhancement of KPR circuits.

The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding.

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~ 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods.

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Single Molecule Study of the Mechanism of Attack of the Human Antimicrobial Peptide LL-37 on E. coli

Evolutionarily conserved anti-microbial peptides (AMPs) are useful prototypes and promising drugs due to increasing antibiotic resistance. We have used single-cell, time-resolved fluorescence microscopy and single-molecule fluorescence microscopy to study the effects of human cathelicidin LL-37 on the E. coli chromosome (visualized through ParB-GFP), RNA polymerase (RNAP), ribosome and cytoplasmic protein Kaede. This enables us to correlate in real time the motion reduction of chromosome and RNAP, the halting of cell growth, and the permeabilization of the cytoplasmic membrane (CM) by LL-37. We have found that LL-37 could change the spatial distribution of ribosome and reduce the motion of chromosomal loci, RNAP, ribosome and Kaede. For chromosome, the apparent diffusion coefficient D decreased by 10 times on both long (∼ min) and short (∼ s) timescales 10 min after injecting 1x MIC (minimum inhibitory concentration) LL-37, larger than those treated with the ATP-depleting drugs NaN3 and CCCP, suggesting LL-37 might have effects on chromosome beyond energy depletion. Besides, some of the sharp clear foci of ParB complex dissolved into broader but dimmer foci. We speculate that LL-37 dissociates the ParB complex by disrupting the protein-protein or protein-DNA interactions. The general ability of a drug to disrupt protein-DNA interactions would be a catastrophe for infectious bacteria. It indicates that LL-37 has multi-target effects and its damage to bacterial cells goes far beyond the membrane permeabilization. This may make it harder for bacteria to gain resistance. A comprehensive understanding of all these effects and how they are interlinked with each other could be of medical significance.

Structural Insight into Interaction between C20 Phenylalanyl Derivative of Tylosin and Ribosomal Tunnel.

Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.

Evidence for Direct Interactions between RNA Polymerase and Ribosomes.

Transcription and translation are coupled in prokaryotes. Coupling originates from close proximity of the RNA polymerase and the ribosome due to their concurrent binding to the same nascent RNA. A similar proximity between RNA polymerase and the ribosome occurs during ribosome biogenesis; when ribosome assembly ensues while the rRNA is being transcribed. Here we report the existence of direct physical interactions between RNA polymerase core enzyme and vacant ribosome in the absence of mediating factors. The interactions appear to be of ionic nature and persist in the presence of an excess of BSA, RNA, or phosphate. The binding of ribosomes to RNA polymerases saturates at a one to one stoichiometry, while that of RNA polymerase to ribosomes saturates at a two to one. The dissociation constant of these complexes is within the physiological concentration range of free RNA polymerase and ribosomes. When bound to ribosomes, the RNA polymerase adopts a conformation similar to that under high salt which appears to be distinct from that of the transcription elongation complex and holoenzyme. Thermus thermophilus RNA polymerase and ribosomes can form complexes with each other as well as mixed‐species complexes with E coli RNA polymerase and ribosomes. Our results suggest that the interface between RNA polymerase and ribosome is conserved and coordinates the interactions between RNA polymerase and ribosome during transcription‐translation coupling and/or during ribosome biogenesis.Support or Funding InformationUCR collaborative seed grant

Role of Protein L25 and Its Contact with Protein L16 in Maintaining the Active State of Escherichia coli Ribosomes in vivo.

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.