Discovery of a gene family critical to wyosine base formation in a subset of phenylalanine-specific transfer RNAs
ABSTRACT A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3'-side of the tRNA(Phe) anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N(1)-methylguanosine (m(1)G(37)) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m(1)G(37) is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNA(Phe)-m(1)G(37) to tRNA(Phe)-yW.
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ABSTRACT: As the molecular adapters between codons and amino acids, transfer-RNAs are pivotal molecules of the genetic code. The coding properties of a tRNA molecule do not reside only in its primary sequence. Posttranscriptional nucleoside modifications, particularly in the anticodon loop, can modify cognate codon recognition, affect aminoacylation properties, or stabilize the codon-anticodon wobble base pairing to prevent ribosomal frameshifting. Despite a wealth of biophysical and structural knowledge of the tRNA modifications themselves, their pathways of biosynthesis had been until recently only partially characterized. This discrepancy was mainly due to the lack of obvious phenotypes for tRNA modification-deficient strains and to the difficulty of the biochemical assays used to detect tRNA modifications. However, the availability of hundreds of whole-genome sequences has allowed the identification of many of these missing tRNA-modification genes. This chapter reviews the methods that were used to identify these genes with a special emphasis on the comparative genomic approaches. Methods that link gene and function but do not rely on sequence homology will be detailed, with examples taken from the tRNA modification field.Methods in Enzymology 02/2007; 425:153-83. DOI:10.1016/S0076-6879(07)25007-4 · 2.19 Impact Factor
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ABSTRACT: A vast number of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a [4Fe-4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in contact with the α-amino and α-carboxylate groups of S-adenosyl-l-methionine (SAM). This binding mode facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, resulting in a reductive cleavage of SAM to methionine and a 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical then abstracts a target substrate hydrogen atom, initiating a wide variety of radical-based transformations. A subset of radical SAM enzymes contains one or more additional iron-sulfur clusters that are required for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known about the roles of these additional clusters. This review will highlight the most recent advances in the identification and characterization of radical SAM enzymes that harbor auxiliary iron-sulfur clusters. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Copyright © 2015. Published by Elsevier B.V.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2015; 1853(6). DOI:10.1016/j.bbamcr.2015.01.002 · 5.30 Impact Factor
Article: N-Alkylated Guanine Derivatives[Show abstract] [Hide abstract]
ABSTRACT: The synthesis, chemical, physical, biological, spectroscopic and miscellaneous analytical properties of Nalkylguanine derivatives substituted at 1-, 1,N²-, N2-, 3-, 3,N2-, 7-, 7,9- and 9-positions have been surveyed, mainly from the 2003-2009 period. Beyond the synthetic methods, particular emphasis has been given to products of mutagenesis and carcinogenesis, the role of modified fluorescent guanosines (wyosine, wyebutosine), mRNA cap structures and drugs stemming from N-alkylguanines. The review is based on 154 references and contains 64 schemes with 355 numbered structures.Current Organic Chemistry 06/2009; 13(11):1085-1135. DOI:10.2174/138527209788680718 · 2.54 Impact Factor