Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5.
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 molecules 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 degrees (the analog of kcat) = 0.0055 min-1 and Km degrees = 0.22 microM. In the limit of GGD5 saturation, the values were: k2 infinity = 0.012 min-1 and Km infinity = 4.5 microM. 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 degrees and Km infinity values, but was only one-third as active over the entire D5 concentration range. A different D5 isomer was entirely ineffective for SJH.
Department of Biological Sciences, University of Pittsburgh, PA 15260.
J S
JS
Franzen
M
M
Zhang
C L
CL
Peebles
D001483:Base Sequence; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013696:Temperature
D012331:RNA, Fungal; D003576:Electron Transport Complex IV