PubMed · 10523206
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A Liljas. 1999-09-24. Function is structure.. https://doi.org/10.1126/science.285.5436.2077
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To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA(Leu) with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5' of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5' to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson-Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.
The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1. 8 mg of purified RNA from a 1 micromol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.
A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G.U wobble base pair formed between a (15)N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin-duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of approximately 20 microM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem-loop of Escherichia coli tRNA(Phe) and suggests that this hairpin has a 3 nt loop.