ABSTRACT
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
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
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.
Subject(s)
Molecular Mimicry , Peptides/chemistry , RNA, Transfer, Amino Acyl/chemical synthesis , RNA/chemistry , Amino Acid Sequence , Base Sequence , Hydrolysis , Protein Biosynthesis , RNA, Transfer, Amino Acyl/chemistryABSTRACT
Labeling of RNA with site-specific fluorine atoms has become straightforward in recent years and in particular has become an integrated part of engineered functional RNA with therapeutic potential, e.g. for siRNA technologies. Here, we demonstrate that temperature-dependent one-dimensional (19)F NMR spectroscopy of oligoribonucleotides is a powerful tool to obtain direct information on the conformational behavior. The approach is particularly useful for the quantification of monomolecular vs bimolecular secondary structure equilibria.
Subject(s)
Fluorine Radioisotopes/chemistry , Magnetic Resonance Spectroscopy/methods , RNA/chemistry , Hydrogen-Ion Concentration , Models, Chemical , Nucleic Acid Conformation , Nucleic Acids/chemistry , Oligoribonucleotides/chemistry , Protein Structure, Secondary , RNA, Small Interfering/metabolism , Temperature , Thermodynamics , Ultraviolet RaysABSTRACT
(19)F-NMR spectroscopy is shown to be a useful tool for monitoring hybridization of nucleic acids.
Subject(s)
Fluorine/chemistry , Nuclear Magnetic Resonance, Biomolecular , Oligonucleotide Probes/chemistry , RNA/chemistry , Isotopes , Nucleic Acid Conformation , TemperatureABSTRACT
By using a structure-based fluorescence spectroscopic approach, we have examined the folding of an adenine-responsive riboswitch that regulates translation initiation. We observed adaptive recognition of the ligand for the aptamer domain of adenosine deaminase (add) mRNA from Vibrio vulnificus, and revealed pronounced conformational changes even in the preorganized loop-loop region that is distant from the binding site. Importantly, the full-length riboswitch domain, which has a potential translational repressor stem is able to form a binary complex with adenine, and does not act as a folding trap to inhibit binding. The aptamer that is extended by the expression platform therefore remains fully responsive to its ligand; this is in contrast to the previously investigated pbuE A-riboswitch, which becomes trapped in a nonresponsive terminator fold. Consequently, the latter must employ complex response mechanisms, such as operating in kinetic-control mode or using transcriptional pausing, to provide time for the aptamer portion to fold and to bind. The different behavior of the riboswitches can be rationalized by their distinct sequence interface between the aptamer and expression platform. For the add A-riboswitch, our data suggest a thermodynamically driven response mechanism.