ABSTRACT
HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.
Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Thermodynamics , Calorimetry , HIV Reverse Transcriptase/chemistry , Nucleotides/chemistry , Nucleotides/metabolism , Polymerization , Reverse Transcriptase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.
Subject(s)
DNA, Catalytic/metabolism , Drug Resistance, Bacterial , Macrolides/pharmacology , Peptides , RNA Stability , RNA, Transfer, Amino Acyl/chemical synthesis , RNA, Transfer, Val/chemistry , Anti-Bacterial Agents/pharmacology , Base Sequence , Escherichia coli , Mass Spectrometry , Models, Molecular , Nucleic Acid Conformation , Phenol/chemistry , Phosphorylation , RNA, Bacterial/metabolism , RNA, Transfer, Val/chemical synthesis , RNA, Transfer, Val/isolation & purification , RNA, Transfer, Val/metabolismABSTRACT
RNA-peptide conjugates that mimic amino acid-charged tRNAs and peptidyl-tRNAs are of high importance for structural and functional investigations of ribosomal complexes. Here, we present the synthesis of glycyl-, alanyl-, and isoleucyladenosine modified solid supports that are eligible for the synthesis of stable 3'-aminoacyl- and 3'-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work significantly expands the range of accessible peptidyl-tRNA mimics for ribosomal studies.
Subject(s)
Polystyrenes/chemistry , RNA, Transfer, Amino Acyl/chemical synthesis , RNA/chemistry , Adenosine/chemistry , Alanine/chemistry , Glycine/chemistry , Hydrolysis , Isoleucine/chemistry , Peptides/chemistry , RNA, Transfer, Amino Acyl/chemistry , Ribosomes/metabolism , Solid-Phase Synthesis TechniquesSubject(s)
Azides/chemistry , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Animals , Azides/chemical synthesis , Base Sequence , Binding Sites , Cell Line , Deoxyadenosines/chemical synthesis , Deoxyadenosines/chemistry , Deoxyadenosines/genetics , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemical synthesis , Deoxyuridine/chemistry , RNA, Small Interfering/chemical synthesis , Substrate SpecificityABSTRACT
The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.
Subject(s)
Peptides/chemistry , RNA, Transfer/chemistry , Base Sequence , DNA, Catalytic/metabolism , Hydrolysis , Molecular Sequence Data , RNA Ligase (ATP)/metabolism , RNA, Transfer/metabolismABSTRACT
The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.
Subject(s)
Platyhelminths/growth & development , Regeneration/physiology , Stem Cells/cytology , Turbellaria/growth & development , Animals , Cell Differentiation/physiology , Helminth Proteins/genetics , Helminth Proteins/metabolism , Homeostasis , Immunohistochemistry , Microscopy, Electron , Phylogeny , Platyhelminths/physiology , RNA Interference , Stem Cells/metabolism , Tail/physiologyABSTRACT
Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.
