Pseudouridine synthetase Pus1 of Saccharomyces cerevisiae: kinetic characterisation, tRNA structural requirement and real-time analysis of its complex with tRNA.
Pseudouridine synthetase Pus1 from Saccharomyces cerevisiae is a multisite-specific enzyme that catalyses the formation of pseudouridine residues at different positions in several tRNA transcripts. Recombinant Pus1, tagged with six histidine residues at its N terminus was expressed in Escherichia coli and purified. Transcripts of yeast tRNAValand intronless yeast tRNAIlewere used as substrates to measure pseudouridine formation at position 27. The catalytic parameters Kmand kcatfor tRNAValand tRNAIlewere 420(+/-100) nM and 0.4(+/-0.1) min-1, 740(+/-100) nM and 0.5(+/-0.1) min-1, respectively. Pus1 possesses a general affinity for tRNA, irrespective of whether they are substrates. Its equilibrium dissociation constant ranges from 15 nM for the substrate yeast tRNAValand non-substrate yeast intronless tRNAPhe, to 150 nM for the substrate yeast intronless tRNAIle. The difference in the affinity for the different tRNA species is not reflected in the specific activity of the enzyme, indicating that the binding of Pus1 to tRNA is not the kinetically limiting step. The importance of tertiary base-pairs was investigated with several variants of yeast tRNAs. Although dispensable for activity, both the presence of a D-stem-loop and the presence of a G26.A44 base-pair, near the target uridine U27, are important elements for Pus1 tRNA high affinity recognition. The presence of a G26.A44 base-pair in tRNA increases its association constant rate with Pus1 (ka) by a factor of approximately 100, resulting in a decrease of the overall equilibrium dissociation constant (Kd). The dissociation rate (kd) is the same, independent of the presence of a G26.A44 base-pair in the tRNA. A model describing the interaction of Pus1 with tRNA is proposed.
Arluison
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Laboratoire d'Enzymologie et Biochimie Structurales du CNRS, 1 avenue de la Terrasse, Gif-sur-Yvette Cedex, 91198, France.
Buckle
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Grosjean
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D020029:Base Pairing; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000737:chemistry / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014158:Transcription, Genetic
D012349:RNA, Transfer, Asp; D012355:RNA, Transfer, Ile; D012366:RNA, Transfer, Val; D011994:Recombinant Proteins; D012343:RNA, Transfer; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase