Functional effects of exchanging domains and linkers in transcription regulators

转录调节因子中交换域和连接子的功能效应

• 批准号: 7914123
• 负责人: LISKIN SWINT-KRUSE
• 金额: $ 26.2万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7914123
• 关键词: Address; Affect; Affinity; Agreement; Algorithms; Amino Acid Substitution; Amino Acids; Binding; Binding Sites; Bioinformatics; Biological Assay; Cartoons; Cations; Chimera organism; Chimeric Proteins; Communication; DNA; DNA Binding; DNA Binding Domain; Data; Data Set; Databases; Engineering; Escherichia coli; Family; Family Leave; Family member; Genetic Recombination; Genetic Transcription; Genomics; Goals; Gray unit of radiation dose; Homologous Gene; Human Genome Project; In Vitro; Knowledge; Left; Ligand Binding; Ligands; Link; Location; Measurement; Measures; Mediating; Monitor; Mutagenesis; N-terminal; Oranges; Outcome; Positioning Attribute; Protein Binding; Protein Region; Proteins; Relative (related person); Repression; Research Personnel; Role; Screening procedure; Sequence Analysis; Specificity; Structure; Tertiary Protein Structure; Testing; Transcriptional Regulation; Transplantation; Tweens; Variant; Work; design; dimer; functional gain; gene repression; improved; in vivo; multidisciplinary; novel; pathogen; programs; research study; response

DESCRIPTION (provided by applicant): Protein homologues often have common functions, with variation evolving via amino acid substitutions at specificity determinants (SDs). These residues may be located in binding sites or other "long-range" regions of the protein. Predicting SDs is a current target of bioinformatics sequence analyses. Our long- term goal is to experimentally identify long-range SDs in the Lacl/GaIR family; the proposed work focuses on SDs in the linker that connects the DMA-binding domain to the regulatory domain. Hypotheses are derived from 4 different prediction strategies, which are only in partial agreement with each other. The common function of the Lacl/GaIR proteins - transcription control - allows "moderate through-put" assays of function so that all predictions may be compared. We will test hypotheses in multiple homologues to address the question: Do homologues utilize all SDs available to the common fold (a frequent assumption of prediction algorithms) or do different functions require only a subset of potential SDs? Further, the aspect of function affected by changing an SD cannot yet be reliably predicted, nor is it clear whether a functional change for one SD is the same in all homologues. Our experiments will monitor different aspects of Lacl/GaIR function, including DMA specificity, DNA affinity, and allosteric response to binding regulatory effector molecules. Proposed experiments utilize chimeras comprising the Lacl DMA-binding domain and regulatory domains from E. coli paralogues. Linkers come from Lacl or paralogues. Since each naturally-occurring Lacl/GaIR protein recognizes a different DNA ligand, the common DNA-binding domain allows us to more easily parse functional contributions from binding site and long-range SDs. By definition, making an amino acid substitu- tion at an SD will change function. We will use in vivo repression/response to effector and in vitro thermody- namic measurements of affinity/allosteric response to characterize the chimeras and potential SD variants. Specificity for alternative DNA ligands will be determined. Experiments are designed to answer the following questions: Aim 1: Can one linker facilitate allosteric communication with a variety of regulatory domains? [or do altered linker SD interactions abolish this function?] Aim 2: What are the functional contributions from specific positions in the linker? Aim 3: Can knowledge of SDs [in one linker] be used to transplant lacO1 DNA-binding to other linkers? Results will: (1) Identify the locations of SDs and determine if they make similar functional contributions to several homologues; (2) Yield a list of empirical rules for creating novel Lacl/GaIR proteins with biotechnological utility via domain recombination; and (3) Test the current prediction algorithms and generate a new sequence/function database for improving predictions. This work will facilitate expanded use of data generated by the Human Genome Project.

DESCRIPTION (provided by applicant): Protein homologues often have common functions, with variation evolving via amino acid substitutions at specificity determinants (SDs). These residues may be located in binding sites or other "long-range" regions of the protein. Predicting SDs is a current target of bioinformatics sequence analyses. Our long- term goal is to experimentally identify long-range SDs in the Lacl/GaIR family; the proposed work focuses on SDs in the linker that connects the DMA-binding domain to the regulatory domain. Hypotheses are derived from 4 different prediction strategies, which are only in partial agreement with each other. The common function of the Lacl/GaIR proteins - transcription control - allows "moderate through-put" assays of function so that all predictions may be compared. We will test hypotheses in multiple homologues to address the question: Do homologues utilize all SDs available to the common fold (a frequent assumption of prediction algorithms) or do different functions require only a subset of potential SDs? Further, the aspect of function affected by changing an SD cannot yet be reliably predicted, nor is it clear whether a functional change for one SD is the same in all homologues. Our experiments will monitor different aspects of Lacl/GaIR function, including DMA specificity, DNA affinity, and allosteric response to binding regulatory effector molecules. Proposed experiments utilize chimeras comprising the Lacl DMA-binding domain and regulatory domains from E. coli paralogues. Linkers come from Lacl or paralogues. Since each naturally-occurring Lacl/GaIR protein recognizes a different DNA ligand, the common DNA-binding domain allows us to more easily parse functional contributions from binding site and long-range SDs. By definition, making an amino acid substitu- tion at an SD will change function. We will use in vivo repression/response to effector and in vitro thermody- namic measurements of affinity/allosteric response to characterize the chimeras and potential SD variants. Specificity for alternative DNA ligands will be determined. Experiments are designed to answer the following questions: Aim 1: Can one linker facilitate allosteric communication with a variety of regulatory domains? [or do altered linker SD interactions abolish this function?] Aim 2: What are the functional contributions from specific positions in the linker? Aim 3: Can knowledge of SDs [in one linker] be used to transplant lacO1 DNA-binding to other linkers? Results will: (1) Identify the locations of SDs and determine if they make similar functional contributions to several homologues; (2) Yield a list of empirical rules for creating novel Lacl/GaIR proteins with biotechnological utility via domain recombination; and (3) Test the current prediction algorithms and generate a new sequence/function database for improving predictions. This work will facilitate expanded use of data generated by the Human Genome Project.