Comparative and functional anatomy of group II catalytic introns — a review

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

“…The solvent accessibility is corroborated by severe line broadening of the imino resonances for both nucleobases as observed by NMR. Only the phylogenetically conserved [10,32] wobble pair 5' of A20 forms a rigid base pair in solution, whereas the G7-U21 is poorer defined.…”

Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing

“…Group II introns are comprised of a series of six helical domains, referred to as domains 1-6+ Deletion analysis has demonstrated that, of these domains, only domains 1 and 5 are absolutely required for catalysis (Koch et al+, 1992)+ Nonetheless, a few highly conserved residues are located outside of those domains+ When group IIA and group IIB introns are compared, four conserved residues are seen outside of domains 1 and 5 (Michel et al+, 1989)+ Given their high degree of conservation, it is likely that these residues are important for one or both steps of splicing+ Two of these residues are located in the joining region that separates the 59 exon from domain 1, and two are located in the joining region that separates domain 2 from domain 3 [J(23)]+ The second pair of residues, a conserved GA dinucleotide (i+e+, G588 A589) located in J(23), were the subject of this investigation+…”

Complete 5′ and 3′ end maturation of group II intron-containing tRNA precursors

“…We selected three representative examples from the five chloroplast group II intron-containing tRNA genes+ In two of these genes, introns interrupt the anticodon loop; these are the subgroup IIA introns in trnA(UGC) and in trnV(UAC)+ The latter is somewhat atypical in not having a bulging A in the intron domain six (Learn et al+, 1992) suggestive of an alternative hydrolytic splicing pathway+ The third example, trnG(UCC), has a subgroup IIB intron within the stem of the DHU domain (Fig+ 1)+ Thus, in intron-containing precursors, the formation of functional tRNA structures might be compromised by the presence of these introns that are of 811, 598, and 680 nt in length, respectively (tRNA Ala (UGC), Vogel et al+, 1998; tRNA Val (UAC), Zurawski & Clegg, 1984; tRNA Gly (UCC), J+ Vogel, unpubl+)+ Additionally, intimate exon binding site-intron binding site (EBS-IBS) interactions between tRNA and intron domains might further decrease the probability of correct recognition by various processing factors (Fig+ 1; Michel et al+, 1989)+ Primer extension of oligonucleotides localized within the intron region of trnG and trnA nevertheless yielded a prominent termination signal at a position equivalent to the 59 matured tRNA (Fig+ 2), at Ϫ133 and Ϫ103 nt respectively+ Thus, the majority of intron-containing precursor RNAs is correctly 59 processed+ This result is in conflict with a previous report that had suggested tRNA 59-end maturation as a step that takes place after splicing (Delp et al+, 1991)+ However, the lack of introncontaining amplification products in that study can be easily explained by the fact that shorter products of PCR are much more efficiently produced than longer ones and that no primers binding within the introns were used+…”

“…Primary and secondary structure of tRNA(Gly) UCC and tRNA(Ala) UGC from barley chloroplasts+ Insertion sites of group II introns within the anticodon loop 2 nt adjacent to the terminal anticodon nucleotide, or within the D stem, are indicated by filled arrows+ The nucleotides involved in the interaction between EBS and IBS, and d-d9 are shown together with a corresponding part of the group II intron (Michel et al+, 1989; J+ Vogel, unpubl+)+ FIGURE 2. Primer extension analysis of precursors for tRNA Gly (UCC) and tRNA Ala (UGC)+ A: Localization of 59 exon, intron, primers, and products of reverse transcription+ B: Products of primer extension with RNA from green (G) or white (W) albostrians plastids+ A control reaction without RNA was included (Ϫ)+ Unincorporated primer is labeled P+ The origin of electrophoresis is marked by S+ Molecular weight marker bands (in base pairs) are shown+ C: Protocol of tRNA precursor circularization followed by reverse transcription and PCR+ Sizes of expected PCR products are given for tRNA Ala (UGC)+ D: Electrophoretic separation of products obtained from 59-39 ligated cDNAs by PCR using primers for tRNA Ala (UGC)+ with the previously reported nonsplicing phenotype for trnA+ Although exclusively intron-containing precursors to tRNA Ala (UGC) are present in these plastids, the RNase P-mediated 59 maturation seems to proceed efficiently+ Consequently, neither interruption of the anticodon or DHU domain nor putative EBS-IBS interactions blocked recognition by RNase P+ Our data provide a first set of tRNA structural elements that are dispensable for catalytic activity of chloroplast RNAse P+ The smallest tRNA-derived substrate that can still be cleaved by E. coli RNase P consists of only the amino acid acceptor stem and the T domain (McClain et al+, 1987)+ The chloroplast enzyme substantially differs from the bacterial one in that it does not utilize a ribozyme-type cleavage mechanism (Thomas et al+, 2000)+ As seen for its counterpart from E. coli, this enzyme depends on neither the anticodon nor the DHU domain to fulfill its function despite a composition that sets it apart from all other known examples of RNase P+ Circularization of wild-type RNA followed by amplification using two primers located within the respective introns was employed to characterize tRNA precursor 59 and 39 ends at the sequence level (Fig+ 2C)+ The result of such an amplification is shown for tRNA Ala (UGC) in Figure 2D+ Most of the obtained products were specific as indicated by the major PCR product of 214 bp+ A 141-bp product originating in reverse transcription of intron molecules in lariat form (Vogel et al+, 1997a) was found with RNA from green plants only (Vogel & Börner, in prep+)+ Interestingly, none of the three tRNA species studied gave major amplification products of significantly larger size than expected for the 59-and 39-processed tRNA precursors+ Thus, these cDNAs represent RNAs that have already undergone efficient processing near to or at the mature 59 and 39 termini of the tRNA exons+ However, because of the nature of our PCR approach, we cannot draw any conclusion as to whether they are major or minor processing intermediates+ Following cloning of the PCR products in E. coli, sequence analysis was performed on 28 individual clones for tRNA Ala (UGC), 11 for tRNA Val (UAC), and 34 for tRNA Gly (UCC) ( Tables 1 and 2)+ In all cases, both exons and adjacent intron sequences were identified+ As expected, the 39 end of exon 2 was linked to the 59 end of exon 1+ The diverse sets of nucleotides occasionally present between these ends could either represent precursor-specific sequences (upstream/downstream of exon 1/2) or ...…”