Nucleic Acids Research, 1993, Vol. 21, No. 3 627-634
© 1993
MOLECULAR BIOLOGY |
Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5
Department of Biological Sciences, University of Pittsburgh Pittsburgh, PA 15260, USA
*To whom correspondence should be addressed
Received September 25, 1992. Revised December 21, 1992. Accepted December 21, 1992.
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.
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