ABSTRACT
Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.
Subject(s)
Anti-Bacterial Agents/pharmacology , Macrolides/pharmacology , Protein Biosynthesis/drug effects , Ribosomes/drug effects , Allosteric Regulation , Cell-Free System , Molecular Conformation , Molecular Dynamics Simulation , Ribosomes/geneticsABSTRACT
The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.
Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Endonucleases/metabolism , Gene Editing/methods , HEK293 Cells , Humans , RNA/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismABSTRACT
Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Ketolides/pharmacology , Macrolides/pharmacology , Protein Synthesis Inhibitors/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Bacillus subtilis/drug effects , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Binding Sites/genetics , Cryoelectron Microscopy , Drug Resistance, Microbial/genetics , Erythromycin/chemistry , Erythromycin/pharmacology , Genes, Bacterial , Ketolides/chemistry , Ketolides/pharmacokinetics , Macrolides/chemistry , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Molecular Dynamics Simulation , Mutagenesis, Insertional , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/chemistry , Ribosomes/drug effectsABSTRACT
The increased incidence of bacterial resistance to available antibiotics represents a major global health problem and highlights the need for novel anti-infective therapies. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. AMPs are versatile, have almost unlimited sequence space, and can be tuned for broad-spectrum or specific activity against microorganisms. However, several obstacles remain to be overcome in order to develop AMPs for medical use, such as toxicity, stability, and bacterial resistance. We lack standard experimental procedures for quantifying AMP activity and do not yet have a clear picture of the mechanisms of action of AMPs. The rational design of AMPs can help solve these issues and enable their use as new antimicrobials. Here we provide an overview of the main physicochemical features that can be engineered to achieve enhanced bioactivity and describe current strategies being used to design AMPs.
Subject(s)
Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Peptides/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Chemical Phenomena , Computational Chemistry , Hydrophobic and Hydrophilic Interactions , Mutagenesis, Site-Directed , Peptides/genetics , Peptides/pharmacologyABSTRACT
Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.
Subject(s)
CRISPR-Cas Systems , DNA/chemistry , RNA, Guide, Kinetoplastida/chemistry , Ribonucleases/chemistry , Databases, Protein , Deoxyribonucleases/chemistry , Escherichia coli , Gene Library , Nucleic Acid ConformationABSTRACT
Macrolide antibiotic binding to the ribosome inhibits catalysis of peptide bond formation between specific donor and acceptor substrates. Why particular reactions are problematic for the macrolide-bound ribosome remains unclear. Using comprehensive mutational analysis and biochemical experiments with synthetic substrate analogs, we find that the positive charge of these specific residues and the length of their side chains underlie inefficient peptide bond formation in the macrolide-bound ribosome. Even in the absence of antibiotic, peptide bond formation between these particular donors and acceptors is rather inefficient, suggesting that macrolides magnify a problem present for intrinsically difficult substrates. Our findings emphasize the existence of functional interactions between the nascent protein and the catalytic site of the ribosomal peptidyl transferase center.
Subject(s)
Escherichia coli/drug effects , Macrolides/pharmacology , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/drug effects , Amino Acid Motifs , Base Sequence , Binding Sites , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Macrolides/chemistry , Peptidyl Transferases/genetics , Peptidyl Transferases/metabolism , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Transfer, Amino Acyl/genetics , Ribosomes/metabolism , Static Electricity , Substrate SpecificityABSTRACT
A key mechanism of bacterial resistance to macrolide antibiotics is the dimethylation of a nucleotide in the large ribosomal subunit by erythromycin resistance methyltransferases. The majority of erm genes are expressed only when the antibiotic is present and the erythromycin resistance methyltransferase activity is critical for the survival of bacteria. Although these genes were among the first discovered inducible resistance genes, the molecular basis for their inducibility has remained unknown. Here we show that erythromycin resistance methyltransferase expression reduces cell fitness. Modification of the nucleotide in the ribosomal tunnel skews the cellular proteome by deregulating the expression of a set of proteins. We further demonstrate that aberrant translation of specific proteins results from abnormal interactions of the nascent peptide with the erythromycin resistance methyltransferase-modified ribosomal tunnel. Our findings provide a plausible explanation why erm genes have evolved to be inducible and underscore the importance of nascent peptide recognition by the ribosome for generating a balanced cellular proteome.