Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase

Blocker, Forrest; Mohr, Georg; Conlan, L. H.; Qi, Li; Belfort, Marlene; Lambowitz, Alan M.

doi:10.1261/rna.7181105

Cited by 87 publications

(126 citation statements)

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“…The RTs of group II introns and non-LTR retrotransposons contain seven conserved sequence blocks (RT-1-RT-7) characteristic of all RTs, but differ from retroviral RTs in having an N-terminal extension with conserved sequence block RT-0, as well as additional insertions in the RT and thumb domains, some with conserved structural features in group II intron and non-LTR-retrotransposon RTs (Xiong and Eickbush 1990;Malik et al 1999;Blocker et al 2005). Like group II intron RTs, non-LTR-retrotransposon RTs promote retrotransposition by using a target DNA-primed reverse transcription mechanism in which a cleaved DNA strand is used as a primer for reverse transcription of the element's RNA, and the cDNA initiation site is determined primarily by specific binding of the RNA template rather than by base pairing of a primer, as for retroviral RTs (Luan et al 1993;Zimmerly et al 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Like group II intron RTs, non-LTR-retrotransposon RTs promote retrotransposition by using a target DNA-primed reverse transcription mechanism in which a cleaved DNA strand is used as a primer for reverse transcription of the element's RNA, and the cDNA initiation site is determined primarily by specific binding of the RNA template rather than by base pairing of a primer, as for retroviral RTs (Luan et al 1993;Zimmerly et al 1995). It has been speculated that the N-terminal extension and/or other RT-and thumb-domain insertions in group II intron and non-LTR-retroelement RTs contribute to their distinctive properties, including higher processivity than that of retroviral RTs (Bibillo and Eickbush 2002a) and specific binding of the template RNA for initiation of reverse transcription (Chen and Lambowitz 1997;Bibillo and Eickbush 2002b;Blocker et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase

Gu¹,

Cui²,

Mou³

et al. 2010

RNA

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Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem-loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA's binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA's N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase

Gu¹,

Cui²,

Mou³

et al. 2010

RNA

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“…Our experiments with nicked DNA substrates confirm that bending results from RNP interactions with the 3' exon and not from bottom-strand cleavage itself ( Figure 8). As indicated previously, three-dimensional modeling predicted that the IEP could not interact simultaneously with its target sequences in the 5'-and 3'-exons unless the DNA is bent strongly in the complex (17).…”

Section: Discussionmentioning

confidence: 60%

“…Docking of target DNA onto this three-dimensional model showed that the DNA target sequence is too long for the LtrA protein to bind simultaneously to its interaction sites in the distal 5'-exon and 3'-exon regions unless the DNA is strongly bent in the complex (17). These findings raised the possibility that bendability of the DNA target site might significantly influence the efficiency of the integration reaction.…”

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confidence: 99%

Atomic Force Microscopy Reveals DNA Bending during Group II Intron Ribonucleoprotein Particle Integration into Double-Stranded DNA

et al. 2006

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The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random Escherichia coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time-course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3'-exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5' exon, while engaging in subsequent 3'-exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3' end of the cleaved bottom strand at the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration.Mobile group II introns, which are found in bacterial and organelle genomes, are retroelements that integrate site-specifically ("retrohome") into DNA target sites at high frequency and retrotranspose to ectopic sites that resemble the normal homing site at low frequency (reviewed in refs (1-4)). The integration reactions are mediated by an RNP complex that is formed during RNA splicing and contains the intron-encoded protein (IEP) 1 and the excised intron lariat RNA (3,4). RNPs initiate intron mobility by recognizing relatively long (30−35 bp) DNA target sites, with both the IEP and base pairing of the intron RNA contributing to the recognition of DNA † This work was supported by NIH grant GM37949 and Welch Foundation Grant F-1607 to A.M.L. and by support from the Welch NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript target sequences (5-8). Then, the intron RNA reverse splices into one strand of the DNA and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the recipient genome by host DNA recombination or repair mechanisms (reviewed in refs 3,4). Their very high integration frequencies and target specificity combined with the ability to program insertion into different target sites by modifying the base-pairing sequences of the intron RNA have made it possible to develop mobile group II introns into highly efficient bacterial gene targeting vec...

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“…4 However, previous studies with RNPs assembled in vitro and in vivo also suggest that LtrA binds RNA as a dimer. 7,[19][20][21] Given this conundrum, further analysis is in order. One possibility is that the RIM structure represents a truncated protein from which a sequence that inhibits dimerization has been deleted and that full-length RIM also binds the intron RNA as a monomer.…”

Section: The Monomer-dimer Conundrummentioning

confidence: 99%

Forks in the tracks: Group II introns, spliceosomes, telomeres and beyond

2016

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Group II introns are large catalytic RNAs that form a ribonucleoprotein (RNP) complex by binding to an intron-encoded protein (IEP). The IEP, which facilitates both RNA splicing and intron mobility, has multiple activities including reverse transcriptase. Recent structures of a group II intron RNP complex and of IEPs from diverse bacteria fuel arguments that group II introns are ancestrally related to eukaryotic spliceosomes as well as to telomerase and viruses. Furthermore, recent structural studies of various functional states of the spliceosome allow us to draw parallels between the group II intron RNP and the spliceosome. Here we present an overview of these studies, with an emphasis on the structure of the IEPs in their isolated and RNA-bound states and on their evolutionary relatedness. In addition, we address the conundrum of the free, albeit truncated IEPs forming dimers, whereas the IEP bound to the intron ribozyme is a monomer in the mature RNP. Future studies needed to resolve some of the outstanding issues related to group II intron RNP function and dynamics are also discussed. KEYWORDSCryo-EM; intron-encoded protein; intron RNP structure; Prp8; reverse transcriptase; telomeraseMore than a quarter century ago, evolutionary connections started being inferred between the spliceosome and self-splicing RNAs, specifically the group II intron. 1,2 The spliceosome's catalytic core was proposed to have arisen in an RNA world where nucleic acids are hypothesized to have been the prevailing macromolecular catalysts. Evidence in favor of the relatedness of group II intron RNA and the active center of the modern-day spliceosome mounted over the past decades, based on studies of splicing mechanism, RNA-metal ion coordination and RNA structure (reviewed in ref.3 ). This evidence has revolved around the nature of the RNA catalysts and seems incontrovertible. Now studies of group II intron-encoded proteins (IEPs) add an additional layer of similarity, not only very strikingly to the spliceosome, but also to other extant ribonucleoprotein (RNP) machines like viruses and telomerases. 4,5 RNP and protein structures illuminate RNA-protein interactions While previous efforts have revealed the structure of a group IIA intron at low resolution, 6-8 the recent breakthrough to solve the group IIA intron RNA from Lactococcus lactis in complex with the intron encoded protein (IEP) provides a first nearatomic model of the overall architecture of a group II intron RNP (Fig. 1A). 4 Also, we now have a close-up view of the interaction sites between excised RNA lariat and protein components, giving important insights into functions of the RNP. The IEP, which has multiple roles in splicing and mobility of the intron, has modules with reverse transcriptase (RT) and DNAbinding/endonuclease activities. The RT is subdivided into an RNA-binding region contained within an N-terminal extension (NTE, also referred to as RT0), a fingers-palm domain (RT1-RT7) and a thumb domain (Fig. 1B). The fingers-palm domain act together with the ...

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Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase

Cited by 87 publications

References 56 publications

Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase

Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase

Atomic Force Microscopy Reveals DNA Bending during Group II Intron Ribonucleoprotein Particle Integration into Double-Stranded DNA

Forks in the tracks: Group II introns, spliceosomes, telomeres and beyond

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