Flexibility of the bulge formed between a hairpin ribozyme and deoxy-substrate analogues.
The conformation of the bulge formed between the hairpin ribozyme R derived from (-)sTRSV and noncleavable all-deoxy-substrate analogues dS was studied by photoaffinity labelling. The photolabel deoxy-6-thioinosine was inserted in place of residue G+1 or A-1, located immediately 3' and 5' to the cleavage site, respectively. Upon 335 nm irradiation both substrate analogues were linked to ribozyme at multiple sites. Formation of the R-dS complex is absolutely required for the generation of the crosslinks, since they were detected neither in the absence of Mg2+ nor upon using a ds6I containing 14-mer, unable to interact with the ribozyme. The fraction of ribozyme crosslinked at completion of the reaction increased with increasing analogue concentrations, yielding apparent KD values for the R-dS complex in the range of 5 +/- 2 microM. Multiple crosslinks between ribozyme and each one of the substrate analogues provide clear evidence for a large flexibility of the bulge region.
Vitorino dos Santos
D
D
Institut Jacques Monod, C.N.R.S. Université Paris VII, France.
Fourrey
J L
JL
Favre
A
A
D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D019244:RNA, Satellite; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D016337:RNA, Catalytic; D019244:RNA, Satellite; D012367:RNA, Viral; D013868:Thioinosine; D012313:RNA; D012260:Ribonucleases