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Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4.

ABSTRACT: Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the "target." The identification of direct in vivo targets of activators has been a major challenge. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. Here we perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo [Gal4 interaction defective (GID) mutants]. In contrast to WT Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription, demonstrating the essentiality of the Gal4-Tra1 interaction. In yeast strains expressing a Tra1 GID mutant, binding of Gal4 to the promoter is unexpectedly also diminished, indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the Gal4-Tra1 interaction occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is targeted by other activators, these interactions are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction.

SUBMITTER: Lin L

PROVIDER: S-EPMC3277556 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4.

Lin Ling L Chamberlain Lynn L Zhu Lihua J LJ Green Michael R MR

Proceedings of the National Academy of Sciences of the United States of America 20120123 6

Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the "target." The identification of direct in vivo targets of activators has been a major challenge. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in p ...[more]

PMID: 22308403

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Project description:BACKGROUND:Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication. RESULTS:A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro. CONCLUSIONS:Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.

Dataset Information

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4.

Publications

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4.

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