Biochemical Activities of Group II Intron Encoded Proteins

Group II IEPs, examples of which are shown in Fig. 2, contain several domains associated with different biochemical activities. The RT domain at the N-ter-minus contains conserved sequence blocks RT-1 to -7, characteristic of the fingers and palm of retroviral RTs, along with an additional upstream motif, RT-0 (Xiong and Eickbush 1990; Malik et al. 1999; Zimmerly et al. 2001). The latter is found only in non-LTR-retroelement RTs and may be part of an extended fingers subdomain used for specific binding of template RNA (Chen and Lambowitz 1997; Bibillo and Eickbush 2002). Domain X, which is a site of mutations affecting maturase activity, is located in the position corresponding to the thumb and part of the connection domain of retroviral RTs and likely corresponds to the RT thumb (Mohr et al. 1993; Blocker et al. 2005). The RT and X domains together bind the intron RNA both as a substrate for RNA splicing and as a template for reverse transcription (Kennell et al. 1993; Cui et al. 2004; Blocker et al. 2005). A functional RT active site is not required for RNA splicing, and the RT and maturase activities are readily separated by mutation (Moran et al. 1995; Zimmerly et al. 1995b; Cui et al. 2004).

The C-terminal D and En domains are not required for RNA splicing, but contribute to interaction with the DNA target site during intron mobility. Domain D is required for reverse splicing of the intron RNA into double-strand ed DNA and is thought to recognize specific nucleotide residues in the DNA target, leading to local DNA unwinding (Guo et al. 1997; San Filippo and Lambowitz 2002). Studies with the Ll.LtrB IEP identified two functionally important regions, which were also identified in other IEPs; one of these regions contains a cluster of basic amino acid residues and the other a predicted a-helix (San Filippo and Lambowitz 2002).

The En domain catalyzes second-strand DNA cleavage to generate the primer for target DNA-primed reverse transcription (TPRT) of the intron RNA (Zimmerly et al. 1995a,b; Yang et al. 1996). It contains conserved sequence motifs characteristic of the HNH family of DNA endonucleases, typically interspersed with two pairs of conserved cysteine residues, similar to an arrangement found in the phage T4 endonuclease VII subfamily of HNH endonucleases (Gorbaleyna 1994; Shub et al. 1994; San Filippo and Lambowitz 2002). In the Ll.LtrB IEP, the HNH active site contains a single catalytically essential Mg2+ ion, while the conserved cysteine motifs appear to stabilize the higher-order structure of the domain, but, unlike endonuclease VII, do not contain a coordinated Zn2+ ion, at least in the purified protein (San Filippo and Lambowitz 2002). In both yeast coxl-ll and Ll.LtrB, deletion of the En domain or mutations in critical active-site residues result in loss of RT activity, implying a functional interaction with the RT domain (San Filippo and Lambowitz 2002). Notably, many group II introns, particularly in bacteria, encode proteins lacking the En domain, and at least some of these are mobile, using alternate mechanisms for priming reverse transcription (see below).

Different lineages of mobile group II introns can be distinguished based on their RNA structures and phylogenetic analysis of their RT sequences (Toor et al. 2001; Lambowitz and Zimmerly 2004). Importantly, within these lineages, the IEPs appear to have coevolved with their intron RNAs, with little if any exchange of IEPs between different introns (Toor et al. 2001). As a result, group II IEPs generally splice and mobilize only the intron in which they are encoded and not other group II introns (Saldanha et al. 1999). This situation differs markedly from that for mobile group I introns, which have been invaded by several different types of homing endonucleases that are indiscriminate in their ability to mobilize different introns (Lambowitz and Belfort 1993; Bel-fort et al. 2002).

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