Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities

Liu, Qiaolian; Green, Justin B; Khodadadi, Arbi; Haeberli, Peter; Beigelman, Leonid; Pyle, Anna Marie

doi:10.1006/jmbi.1996.0845

Cited by 36 publications

(41 citation statements)

References 37 publications

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“…3B, lanes 2, 4, and 6), whereas the product derived from the lariat was barely detectable only in the A764C mutant (lane 4), consistent with the low efficiency of cytosine as the branch point nucleotide demonstrated both in vitro and in vivo in other introns (6,26,27). The larger RT-PCR products of ⌬A, A764C, and pU mutants were cloned and sequenced.…”

Section: Detection Of Rmint1 Products In Vivo Bysupporting

confidence: 62%

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

Molina-Sánchez¹,

Barrientos‐Durán²,

Toro

2011

Journal of Biological Chemistry

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Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5 and 3 extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2-5 phosphodiester bond, and tandem, head-to-tail intron copies.Group II introns are large catalytic RNAs with a conserved secondary structure consisting of six domains arranged around a central wheel (1). Group II introns may self-splice via several different pathways (Fig. 1). The main splicing pathway in group II introns involves two sequential trans-esterification reactions. In the first, the 2Ј-hydroxyl of a bulged adenosine near the end of the intron, in domain VI, triggers a nucleophilic attack on the 5Ј splice site. In the second reaction, the 2Ј-5Ј branched product is released as a lariat when the free 3Ј-OH at the end of the 5Ј exon attacks the 3Ј splice junction joining the two exons (2-5). In ai5␥, a yeast mitochondrial group IIB intron, the functional group II intron branch site consists of the bulged adenosine, the wobble pairs (G-U) flanking this residue and, possibly, the purine-enriched sequence in domain VI on the strand opposite the bulged nucleotide (6 -8). Recent studies have reported the presence, within domain I of group IIB introns, of a branch site receptor known as a coordination loop (9), which is required for trans-esterification (8), but these findings have been called into question (10). This coordination loop has been identified as a key element for exon ligation, as it positions the exon and intron binding sequences, the EBS3-IBS3 base pair and the EBS1-IBS1 helix, in an appropriate conformation for reaction (9). There is also an alternative splicing pathway, the first step of which involves hydrolysis. The ligated exons and linear intron are then generated in a standard second step (11-13). An additional excision reaction, in which the intron is excised as a putative circle, has also been proposed for some group II introns (14 -16). In this case, prior release of the 3Ј exon from the precursor molecules, presumably via a transsplicing mechanism, seems to be required for the generation of a putative 2Ј-5Ј ligated circular intron. The origin and function of such intron circles in vivo remains unknown.RmInt1 is a mobile subclass IIB3 intron (17). It has also been assigned to phylogenetic bacterial class D (17, 18) on the basis o...

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Section: Detection Of Rmint1 Products In Vivo Bysupporting

confidence: 62%

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

Molina-Sánchez¹,

Barrientos‐Durán²,

Toro

2011

Journal of Biological Chemistry

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“…However, it is important to note that many differences exist in terms of branch-site recognition and function. [10,33] For example, in yeast, where the two systems are most closely related, the spliceosomal branch-A is not flanked by GU-wobbles, a feature that is highly conserved in group IIB introns. [10] Instead, a conserved spliceosomal seven nucleotide sequence [34] together with a pseudouridine opposite to the branch adenosine are likely to be responsible for the single bulgedout branch A, [35] which must be unpaired for successful splicing.…”

Section: Discussionmentioning

confidence: 99%

“…These findings are well in line with the observation that A20 X mutations with nucleobases of lesser stacking ability [19,39] lead to reduced branching. [33] Metal ions are key players in group II intron ribozymes, not only stabilizing the complicated tertiary structure, but also being directly implicated in catalysis. [8,14,15] Therefore we have put a strong emphasis on the detailed investigation of metal ion binding sites within our structure.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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Group II intron self-splicing is essential for correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism apparently analogous to the one of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5 . The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix being partially stacked between the two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair stacking of the branch-adenosine, but in contrast rather strengthens the interaction with the flanking uridines, as shown by NMR-and fluorescence studies. This means, that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

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“…1,2 Domain 6 (D6) thereby actively takes part in splicing as this domain contains a highly conserved bulged adenosine whose 2'-OH is the nucleophile in the first step of splicing. [3][4][5][6] We have recently solved the NMR solution structure of a minimal but active branch domain 6 from the yeast mitochondrial group II intron Sc.ai5γ ( Figure 1). 7 This so-called D6-27 construct retains all important branching determinants that lie within the branch-domain itself 8 and has been shown to actively trans-splice in vitro.…”

Section: Introductionmentioning

confidence: 99%

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

Erat

Sigel

2007

Inorg. Chem.

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Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 contains a conserved adenosine whose 2'-OH acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long domain 6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5, identifying also five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di-and triphosphate groups is strongest (e.g., log KA,TP = 4.55 ± 0.10) and could be evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 ± 0.06) and thus compete for the free Mg2+ ions in solution having a distinct influence on the individual affinities of the various sites. For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs. ions in solution having a distinct influence on the individual affinities of the various sites.For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs.3

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Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities

Cited by 36 publications

References 37 publications

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

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

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

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Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities

Cited by 36 publications

References 37 publications

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

Relevance of the Branch Point Adenosine, Coordination Loop, and 3′ Exon Binding Site for in Vivo Excision of the Sinorhizobium meliloti Group II Intron RmInt1

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

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg2+Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

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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

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme