RNA-protein interactions of an archaeal homotetrameric splicing endoribonuclease with an exceptional evolutionary history.
The splicing endoribonuclease from Methanococcus jannaschii, a member of a recently defined family of enzymes involved in splicing of archaeal introns and eukaryotic nuclear tRNA introns, was isolated and shown by cross-linking studies to form a homotetramer in solution. A non-cleavable substrate analogue was synthesized by incorporating 2'-deoxyuridines at the two cleavage sites and complexed to the splicing enzyme. The complex was subjected to protein footprinting and the results implicated an RNP1-like sequence and a sequence region immediately N-terminal to a putative leucine zipper in substrate binding. In addition, a histidine residue (His125), positioned within a third RNA binding region, was shown to be involved in catalysis by mutagenesis. The splicing enzyme was localized on the central helix and the two 3 nt bulges of the conserved archaeal 'bulge-helix-bulge' substrate motif by RNA footprinting. Sequence comparison with the dimeric splicing enzyme from Halobacterium volcanii demonstrates that the latter is a tandemly repeated duplication of the former, where alternating segments within each protein half degenerated after the duplication event. Another duplication event, in the eukaryotic domain, produced two different homologues of the M.jannaschii-type enzyme structure. The data provide strong evidence that the tetrameric M.jannaschii enzyme consists of two isologously associated dimers, each similar to one H.volcanii monomer and each consisting of two monomers, where one face of monomer 1 and the opposite face of monomer 2 are involved in RNA binding.
Lykke-Andersen
J
J
RNA Regulation Centre, Institute of Molecular Biology, Copenhagen University, Solvgade 83H, DK-1307 Copenhagen K, Denmark.
Garrett
R A
RA
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004722:Endoribonucleases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006217:Halobacterium / Q000201:enzymology / Q000235:genetics; D007438:Introns; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011485:Protein Binding; D011487:Protein Conformation; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D019843:Archaeal Proteins; D016601:RNA-Binding Proteins; D004722:Endoribonucleases; C109175:splicing endonuclease, Methanococcus