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Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control.

ABSTRACT: Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promoter, where TraR binds to a site centred 45 nucleotides upstream of the transcription start site, and of the traM promoter, where TraR binds a site centred 66 nucleotides upstream. Alteration of 18 residues blocked activity at the traI promoter. Of these, alteration at three positions impaired TraR abundance or DNA binding, leaving 15 residues that are specifically needed for positive control. Of these 15 residues, nine also blocked or reduced activity at the traM promoter, while six had no effect. Amino acid residues required for activation of both promoters probably contact the C-terminal domain of the RNA polymerase alpha subunit, while residues required only for traI promoter activation may contact another RNA polymerase component.

SUBMITTER: Costa ED

PROVIDER: S-EPMC2748755 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control.

Costa Esther D ED Cho Hongbaek H Winans Stephen C SC

Molecular microbiology 20090706 3

Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promote ...[more]

PMID: 19602141

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Project description:Once HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. Host cell factors are essential for these processes, although very few reverse transcription complex binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic translation elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT) for more efficient reverse transcription. Here we report that the surface-exposed acidic residues in the HIV-1 RT thumb domain alpha-J helix and flanking regions are important for interaction with eEF1A. Mutation of surface-exposed acidic thumb domain residues D250, E297, E298, and E300 to arginine resulted in various levels of impairment of the interaction between RT and eEF1A. This indicates that this negatively charged region in the RT thumb domain is important for interaction with the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated with the efficiency of reverse transcription, uncoating, and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the interaction of RT with eEF1A and virus replication in CD4+ T cells without affecting in vitro RT catalytic activity, RT heterodimerization, or RNase H activity. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication.IMPORTANCE HIV-1, like all viruses, requires host cell proteins for its replication. Understanding the mechanisms behind virus-host interactions can lay the foundation for future novel therapeutic developments. Our lab has identified eEF1A as a key HIV-1 RT binding host protein that is important for the reverse transcription of HIV-1 genomic RNA into DNA. Here we identify the first surface-exposed RT residues that underpin interactions with eEF1A. Mutation of one strictly conserved RT residue (E300R) delayed reverse transcription and viral core uncoating and strongly inhibited HIV-1 replication in CD4+ T cells. This study advances the structural and mechanistic detail of the key RT-eEF1A interaction in HIV-1 infection and indicates its importance in uncoating for the first time. This provides a further basis for the development of an RT-eEF1A interaction-inhibiting anti-HIV-1 drug and suggests that the surface-exposed acidic patch of the RT thumb domain may be an attractive drug target.

Dataset Information

Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control.

Publications

Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control.

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