Nucleotide modifications

All classes of RNA transcripts must be processed into mature species. The reactions include several types Nucleolytic cleavage, as in the separation of the mature rRNA species from the primary transcript of RNA polymerase I action Chain extension (non-template-directed), as in the synthesis or regeneration of the common CCA sequence at the 3 end of transfer RNAs or of PolyA at the 3 end of mRNAs and Nucleotide modification, for example, the synthesis of methylated nucleotides in tRNA or rRNA. These reactions are a feature of both prokaryotic and eukaryotic gene expression, and the biological consequences are diverse. For example, modified nucleotides can affect the way in which a tRNA recognizes different codons. 

Izzotti A, Bagnasco M, Camoirano A, et al. 1998. DNA fragmentation, DNA-protein crosslinks, 32P postlabeled nucleotide modifications, and 8-hydroxy-2 -deoxyguanosine in the lung but not in the fiver of rats receiving intratracheal instillations of chromium(VI). Chemoprevention by oral N-acetylcysteine. Mutat Res 400 233-244. 

When tylosin binds to the ribosome, the mycinose extension from C14 of the lactone ring extends down the exit tunnel and interacts with domain II of rRNA (Fig. 4.8 B). Nucleotide modification by N1 methylation of Ec G748 (Hm A841) in... 

Decatur WA, Fommer MJ. RNA-guided nucleotide modification of ribosomal and other RNAs. J. Biol. Chenn 2003 278 695-698. 

The types of nucleoside or nucleotide modifications range from alterations of the base component to changes in the sugar or phosphate moieties. The natural modifications include pseudouridylation (generation of a C-glycoside), methylation. 

A series of eight different non-hydrolysable GDP analogues (112a-h) has been used in an affinity study between the G-protein of the visual photoreceptor, transducin, and GDP. The imidodiphosphate derivative (112e) exhibited good affinity to transducin. This very important heterotrimeric G-protein was shown to be highly restrictive with regard to the structural nucleotide modifications at the pyrophosphate moiety, at the 3 -position of the ribose and at the N position of the purine. ... 

Ans. Capping and splicing occur in the nucleus while most nucleotide modifications generally take place in the cytosol (the region outside the nucleus and other organelles). 

Figure 7.5-4. Nucleotide modifications for use in antisense technology. (A) The first-generation phosphorothioate backbone modification. (B) The second ribonucleotides modified at the 2 hydroxyl by adding a methyl or methoxy-ethyl group. (C) The third-generation modifications involving the complete replacement of phosphodiester linkages as in PNA and MF or the conformational lock as in LNA.

DNAzymes often rely on metal ions for their activity, possibly to compensate for the limited functional diversity of DNA. The role of the metal ions can be either catalytic or structural, but in most cases this remains to be established. To date, the synthetic potential of DNAzymes is rather limited and their main application has been in nucleotide modification. However, since DNAzymes can be selected from large libraries using in vitro evolution and selection methodologies, metallo-DNAzymes for a wide range of synthetically relevant reactions (e.g., C-C bond forming reactions) will undoubtedly become available in the future. 

This is small nucleolar RNA or snoRNA. These RNA species recognize their target sequence by base-pairing and then recruit specialized proteins that perform nucleotide modifications to these RNAs usually 2 O-ribose methylation, base deaminations such as adenine-to-inosine conversions, and the addition of pseudouridines. These modifications are crucial to ribosome biogenesis. 

Helm, M. 2006. Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res. 34 721-33. 

Potential Effects on Transcription or Translation To complement direct polymerase intaaction assays, the potential effects of nonnatural monomeric metaboUles on transcription and translation processes should also be considered. Cell-free recombinant transcriplion/translation kits are commercially available (bacterial or mammalian origin) to enable the characterization of individual monomeric metabolites. In principle, any bad actor chemically modified nucleotide could be identified via alterations to overall RNA yield/integrity. Again, these assays could be conducted in competition-type formats (modified NTP spiked into endogenous NTP pool) mimicking relative exposure ratios in vivo. For translation, synthetic RNA templates could be synthesized that contain the chemical modification in question to see whether the presence of that modification in an RNA template alters overall protein yield. As an early hazard ID-type screen, any nucleotide modifications that behave as chain terminators or have some other inhibitory activity should be readily identified. 

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