RESUMO
Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.
Assuntos
Nucleosídeos , Nucleossomos , DNA/química , RNA Polimerases Dirigidas por DNA/química , Nucleosídeos/química , Nucleotídeos/metabolismoRESUMO
Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.
Assuntos
Antibacterianos/farmacologia , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Sequência de Aminoácidos , Antibacterianos/metabolismo , Carbocianinas/química , Códon , Eritromicina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Inibidores da Síntese de Proteínas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Imagem Individual de MoléculaRESUMO
The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.
Assuntos
Aminoglicosídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Microscopia de Fluorescência , Imagem Molecular/métodos , RNA de Transferência/genética , Ribossomos/metabolismo , Análise de Célula Única/métodosRESUMO
Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.
