TRAINING IN USE OF DMX ELECTRONICS

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There are three classes of recoding: ribosomal frameshifting, stop-codon readthrough, and bypass of internal mRNA region. Recoding instructions may include an RNA structure within the mRNA. These structures can kinetically alter the mRNA passage through the ribosome, can interface with the ribosome directly, and can bind proteins or other co-factors and bring them into place to direct the recoding event. Within the universal code, the UGA codon signals the termination of protein synthesis. This event can be circumvented in three ways, including co-translational insertion of selenocysteine at this triplet (a special case of readthrough). For selenocysteine insertion at stop codons in eukaryotes, there are four requirements: (1) two enzymes essential for the synthesis of the selenocysteine amino acid; (2) a special tRNA; (3) an RNA stem-loop structure, termed the selnocysteine insertion element (SECIS), is required in the 3'-UTR of the mRNAs and; (4) a protein which may act as the homologue to the prokaryotic SelB protein (SBP) is thought to bind the SECIS and program the ribosome for selenocysteine insertion. An NMR structural investigation of this structure will help to understand how an element in the 3'-UTR of a specific mRNA "informs" ribosomes translating that same mRNA that all UGA codons, with the exception of the terminal codon, should specify selenocysteine insertion. Two models of the SECIS secondary structure are available. Both models identify two stems separated by an internal loop, in addition to an apical loop. Phylogeny identifies three consecutive adenosines in loop II, and a run or 4 base-pairs in the "core" region that are essential for the function of SECIS. The base pairing register of stem II differs in the two models by one nucleotide including two tandem G-As which are proposed to be formed using N6-A to the N3-G, and a N7-A to N2-G H-bonds, allowing room for the restricted geometry of the U-U basepairs that flank the tandem. These intriguing helical arrangements are ideal sites for specific recognition of the SelB homologue (SBP) protein for eukaryotic selnocysteine insertion. A43 nucleotide RNA corresponding to stemII/loopII has been produced for NMR studies using runoff T7 transcription in conjunction with double ribozyme cleavage to produce homogeneous RNAs. Simino resonances in the 1D 1H spectra of this RNA lie within the chemical shift range expected for G-A base pairs. This attribute strongly suggests model B to be more consistent with the stemII/loopII region of this SECIS RNA. A smaller RNA has been purified, which corresponds to stemII with a UUCG tetraloop replacing loopII, to simplify the assignment of stem resonances. This RNA has indirectly confirmed the base pairing register to be consistent with model B, by forcing this stem arrangement using the tetra-loop junction, and observing a nearly identical imino spectrum. Homonuclear 2D 1H data have suggested the same base-pairing arrangement of model B, but have been limited by degeneracies in the 1H spectra. Isotopic enrichment of these RNAs will be essential for more complete assignments required for the determination of a high resolution structure. These include complete random isotopic enrichment of RNAs in both 15N and 13C, and selective enrichment of specific nucleotides.