Discovery of Self Splicing Group I Introns

One of the first genomic projects, begun in the late 1970s, included efforts by several groups to deduce the sequences of the mitochondrial (mt) genomes of fungi. These projects were undertaken as it became clear that the expression of mt genes was controlled by both nuclear and other mt factors. To understand the regulatory mechanisms, the mt DNA of respiratory mutants in S. cerevisiae (which are viable under anaerobic conditions) as well as mt suppressor mutant strains were sequenced. One of the most remarkable observations that came from these projects was that mt genes were not contiguous: many open reading frames (ORFs) were interrupted by "inserts". Such a "mosaic" pattern of genes was concurrently discovered in viral and eukaryotic genes. With these discoveries, it became clear that, in such instances, mRNAs must be processed (spliced) to remove the intragenetic sequences (introns) and join together the coding sequences (exons). Importantly, the analysis of many respiratory deficient strains in S. cerevisiae revealed that mt mRNAs were not fully processed leading to truncated, inactive mt proteins.

Sequence analysis of the fungal mt introns showed that they fell into one of two structural classes, groups I and II (for a discussion of group II introns, see Lambowitz et al., this Vol.). Other group I introns were subsequently found in chloroplasts, cyanellar and nuclear genomes in eukaryotes, as well as bacteria and bacteriophages. Group I introns form a characteristic secondary structure, as shown in Fig. la. Biochemical analyses showed that group I introns were self-splicing, requiring only divalent cations and a guanosine co-factor for activity (reviewed in Cech 1990). The group I splicing mechanism consists of two sequential transesterification reactions. In the first step, the in-tron core binds an exogenous guanosine that attacks the 5' splice site (SS) resulting in covalent attachment of the guanosine to the first intron nucleotide. In the second step, the 5' exon attacks the 3' SS, resulting in exon ligation and intron release (Cech 1990). The 5' and 3' SS are defined by pairing to an intron structure called the internal guide sequence, forming the PI and P10 helices, respectively (Fig. la,b).

The group I intron catalytic core is made up of two extended helices called domains; the P4-P6 domain is formed by coaxial stacking of P5, P4, P6, and P6a helices while the P3-P9 domain is formed by stacking of P8, P3, P7, and P9 (Michel and Westhof 1990; Golden et al. 1998; Adams et al. 2004; Fig. la). Precise packing of the two domains via tertiary interactions forms the intron catalytic center that is capable of binding guanosine and the SS containing PI

and P10 helices. Interconnecting peripheral structures surround the intron core and stabilize the active conformation (Lehnert et al. 1996; Pan and Woodson 1999; Engelhardt et al. 2000; Fig. la). Folding of group I introns into an active conformation is often very slow due to long-lived, kinetically trapped intermediates (Woodson 2000). In vivo, folding can be accelerated by the binding of protein cofactors (Schroeder et al. 2002).

Fig. 1. a Group I intron conserved secondary structure. Exon and intron sequences are represented by thick and thin lines, respectively. In this diagram, the P2, P5a, b and P9.1b helices represent optional peripheral folding elements outside of the catalytic core, b Comparison of DNA target site sequences and the pre-RNA secondary structure around the 5' and 3' splice sites. For the DNA substrate, arrows indicate endonuclease cleavage sites used to initiate a homing event. For the RNA sequence, upper- and lowercase letters indicate intron and exon sequences, respectively. Arrows point to the splice sites. The sequences represent the I-Anil endonuclease/maturase substrates. Gray bases indicate the identical residues between the DNA site and P1/P10 pseudoknot. c Topology of I-Anil. The DNA-binding surface is composed of antiparallel p-sheets shown as black arrows. a-Helices are shown as gray cylinders and the LAGLIDADG helices are shown as gray circles. This "top" view emphasizes the non-DNA-binding surface of I-Anil that plays a major role in matu-rase activity. C and N, C- and N-termini, respectively

Fig. 1. a Group I intron conserved secondary structure. Exon and intron sequences are represented by thick and thin lines, respectively. In this diagram, the P2, P5a, b and P9.1b helices represent optional peripheral folding elements outside of the catalytic core, b Comparison of DNA target site sequences and the pre-RNA secondary structure around the 5' and 3' splice sites. For the DNA substrate, arrows indicate endonuclease cleavage sites used to initiate a homing event. For the RNA sequence, upper- and lowercase letters indicate intron and exon sequences, respectively. Arrows point to the splice sites. The sequences represent the I-Anil endonuclease/maturase substrates. Gray bases indicate the identical residues between the DNA site and P1/P10 pseudoknot. c Topology of I-Anil. The DNA-binding surface is composed of antiparallel p-sheets shown as black arrows. a-Helices are shown as gray cylinders and the LAGLIDADG helices are shown as gray circles. This "top" view emphasizes the non-DNA-binding surface of I-Anil that plays a major role in matu-rase activity. C and N, C- and N-termini, respectively

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