Kinetic analysis of the 5′ splice junction hydrolysis of a group II intron promoted by domain 5

• Published in: Nucleic acids research Vol. 21; no. 3; pp. 627 - 634
• Main Authors: Franzen, James S., Zhang, Mincheng, Peebles, Craig L.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.02.1993
• Subjects: Base Sequence; Biological and medical sciences; Electron Transport Complex IV - genetics; Electron Transport Complex IV - metabolism; Fundamental and applied biological sciences. Psychology; Hydrolysis; Introns; Kinetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nucleic Acid Conformation; RNA Splicing; RNA, Fungal - chemistry; RNA, Fungal - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Temperature; Transcription. Transcription factor. Splicing. Rna processing; Hydrolysis; Temperature; Messenger RNA; Splicing; Intron; Environmental factor; Junction; Intermolecular reaction; Kinetic parameter; In vitro; Precursor
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/21.3.627

The 5′ splice junction (5′SJ) of Group II intron transcripts is subject to a specific hydrolysis reaction (SJH). This reaction occurs either within a single transcript containing intron sequences through domain 5 (D5) or by cooperation of two separate transcripts, one bearing the 5′SJ and another contributing D5 (1). In this report we describe the latter reaction in terms of its kinetic parameters. A minimal D5 RNA of 36 nts (GGD5) was sufficient to promote SJH of a second transcript containing the 5′ exon plus intron domains 1, 2, and 3 (E1:123). Equimolar production of two RNAs, the 5′ exon (E1) and an intron fragment containing domains 1, 2, and 3(123) was observed. The kinetic coefficients were evaluated by an excess GGD5 approach. The apparent Km was complex, varying with GGD5 concentration. This behavior indicates heterogeneity in E1:123 with respect to GGD5 binding. The binding heterogeneity may result from formation of E1:123 dimers or from nicks in some moiecules of each E1:123 preparation. The heterogeneity was always evident, but to a variable degree, regardless of the procedure by which E1:123 was isolated. The system may be described in terms of parameters analogous to Kcat and Km. At infinite dilution of GGD5, the characterizing values were: K2° (the analog of Kcat) = 0.0055 min-1 and Km° = 0.22 μM. In the limit of GGD5 saturation, the values were: K2∞ = 0.012 min-1 and Km∞ = 4.5 μM. A natural variant D5, representing the sequence from intron 1 of the yeast cytochrome-b gene, was also functional in SJH. This GGD5b1 was governed by similar Km° and Km∞ values, but was only one-third as active over the entire D5 concentration range. A different D5 isomer was entirety ineffective for SJH.