THIONUCLEOBASES AS INTRINSIC PHOTOAFFINITY PROBES OF NUCLEIC-ACID STRUCTURE AND NUCLEIC-ACID PROTEIN INTERACTIONS

In the past few years thionucleobases have been extensively used as in trinsic photolabels to probe the structure in solution of folded RNA m olecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non- natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine ab sorb light at wavelengths longer than 320 nm and, thus, can be selecti vely photoactivated. Synthetic or enzymatic procedures have been estab lished, allowing the random or site-specific incorporation of thionucl eotide(s) within a RNA (DNA) chain which, in most cases, retains unalt ered structural and biological properties. Owing to the high photoreac tivity of their triplet state (intersystem yield close to unity), 4-th iouracil and 4-thiothymine derivatives exhibit a high photocrosslinkin g ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide m ixtures and in dinucleotides, the main photochemical pathway was ident ified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose stru cture could be characterized. Depending on the mutual orientation of t hese bonds in the thietanes, their subsequent dark rearrangement yield ed, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A si milar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher r eactivity of thymine derived accepters can be explained by an addition al pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleoside s inserted in nucleic acid chains has been used to probe RNA-RNA conta cts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional in teractions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained with in small endonucleolytic ribozymes in solution led to a tertiary folde d pseudo-knot structure for the HDV ribozyme and allowed the construct ion of a Y form of a hammerhead ribozyme, which revealed to be in clos e agreement with the structure observed in crystals. Thionucleosides i ncorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is kno wn about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defi ned nucleic acid site and applied to various systems (replisome, splic eosome, transcription complexes and ribosomes). (C) 1998 Elsevier Scie nce S.A.