Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro

Nguyen, Phong Quoc; Conesa, Christine; Rabut, Elise; Bragagnolo, Gabriel; Gouzerh, Célia; Fernández‐Tornero, Carlos; Lesage, Pascale; Reguera, Juan; Acker, Joël

doi:10.1016/j.jbc.2021.101093

Cited by 6 publications

(17 citation statements)

References 68 publications

(104 reference statements)

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“…Our maps show no density for the structured N-terminal half of IN1, containing functional domains for integration of the Ty1 cDNA. This suggests that the ∼230 residue linker connecting the N-terminal half to the C-terminus of IN1 (Figure 2B), which is disordered 19 , provides significant flexibility between the Pol III-tethering and DNA-integration modules of IN1. Ty1 integration occurs within the first to the third nucleosomes upstream of Pol III and, thus, a flexible linker is advantageous to reach varying distances while keeping strong tethering on genome-attached Pol III.…”

Section: Discussionmentioning

confidence: 99%

“…A third piece of additional density (Figure 1, black star) exhibits a globular shape and localizes next to the Pol III stalk, both contacting a peripheral loop (residues 35-51) of subunit C17 and an a-helix (residues [15][16][17][18][19][20] in the tip domain of subunit C25 (Figure 3A). This density also contacts the nearby dock domain of subunit C160.…”

Section: Cryo-em Structures Of Pol Iii-in1 Complexesmentioning

confidence: 99%

“…Cells were harvested at 1100g for 15 min at room temperature, washed with PBS 1X and weighed. Purification of IN1 was performed following described procedures 19 .…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…To gain structural insights into IN1 tethering on Pol III, we first formed an in vitro complex between recombinant IN1 produced in bacteria 19 and the Pol III enzyme isolated directly from yeast 20 . The two species interacted in a 1:1 stoichiometry as shown by native gel electrophoresis (Figure S1A), demonstrating a direct interaction between the two enzymes.…”

Section: Cryo-em Structures Of Pol Iii-in1 Complexesmentioning

confidence: 99%

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Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

Nguyen

Huecas

Asif-Laidin

et al. 2022

Preprint

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The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.

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Section: Discussionmentioning

confidence: 99%

Section: Cryo-em Structures Of Pol Iii-in1 Complexesmentioning

confidence: 99%

“…Cells were harvested at 1100g for 15 min at room temperature, washed with PBS 1X and weighed. Purification of IN1 was performed following described procedures 19 .…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Cryo-em Structures Of Pol Iii-in1 Complexesmentioning

confidence: 99%

See 2 more Smart Citations

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

Nguyen

Huecas

Asif-Laidin

et al. 2022

Preprint

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“…Ty1 IN is a 71-kDa protein of 635 amino acid residues, produced from a rather complex semi-ordered Gag-Pol proteolytic cleavage pathway (23). Ty1 IN harbours the conserved three domain structure of retroviral integrases: the Nterminal oligomerization domain (NTD) containing a conserved HHCC zinc-binding motif, the central catalytic core domain (CCD) with the invariant DD 35 E motif and the less conserved C-terminal domain (CTD), which, in the case of Ty1, is particularly large and ends with an intrinsically disordered region (24).…”

Section: Introductionmentioning

confidence: 99%

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

Barkova

Adhya

Conesa

et al. 2022

Preprint

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Background. Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN).Results. Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. ConclusionOur study highlights an important role of CK2 in the regulation of Ty1 retrotransposition. We provide the first evidence that Ty1 IN is post-translationally modified in vivo, as observed for retroviral INs, and demonstrate that CK2 strongly represses Ty1 mobility by inhibiting Ty1 transcription. The proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

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Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

et al. 2023

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The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. We report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Binding to IN1 associates with allosteric changes in Pol III that may affect its transcriptional activity. The C-terminal domain of subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore, providing evidence for a two-metal mechanism during RNA cleavage. Additionally, ordering next to C11 of an N-terminal portion from subunit C53 may explain the connection between these subunits during termination and reinitiation. Deletion of the C53 N-terminal region leads to reduced chromatin association of Pol III and IN1, and a major fall in Ty1 integration events. Our data support a model in which IN1 binding induces a Pol III configuration that may favor its retention on chromatin, thereby improving the likelihood of Ty1 integration.

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Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro

Abstract: Ty1 integrase is composed of an active N-terminal domain and a large disordered Cterminal module dispensable for its activity in vitro

Cited by 6 publications

References 68 publications

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

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