Function discovery platform for transcriptional regulators

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Bacterial Transcriptional Regulators (TRs) control transcription by positively or negatively regulating protein expression. The binding of TRs to the DNA is determined by the concentration of an Effector, which is often a product of the biochemical pathway. Binding of the Effector to the TR generates a binary complex responsible to promote or repress gene transcription. The figure depicts an example of TR mediated gene repression.

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The large-scale genome sequencing efforts by the DOE/Joint Genome Institute and others has resulted in sequences for close to 10,000 bacterial genomes. The work’s most profound outcome is the revelation of how little is known about the functions of a very large fraction of genes in every organism. More sequencing alone will not solve this problem. Even in E. coli, the most comprehensively studied organism, a complete functional description cannot be assigned to over 50% of the protein-encoding genes. The genes with unknown function are divided into three main classes: 1) genes encoding for non-catalytic proteins (such as structural proteins and transcriptional factors), 2) genes encoding for enzymes catalyzing biochemical reactions in pathways that have not yet been discovered, and 3) genes encoding for enzymes catalyzing reactions in known pathways (orphan enzymes). To derive phenotype (observed properties resulting the interaction of the genetic makeup with environment) from genotype (genetic makeup of the organism), scientists must have a complete understanding of cellular function. This will require a detailed understanding of each individual gene/protein function (specific activity), their involvement in metabolic pathways or networks, and the overall genetic and biochemical regulation governing these pathways and networks. The presence of thousands of conserved genes of unknown function means that a large fraction of the biochemistry and physiology cannot be inferred simply by analyzing genome sequences. Solving this problem is one of the great challenges that biochemists face. Understanding how the information contained in genes manifests as an organism’s traits, the phenotypes, remains one of the main challenges for systems biology. Laboratory scientists addressed this issue in a paper published in BMC Genomics.

The interdisciplinary Lab team designed, created, and validated a hybrid platform for the discovery of biological function of proteins with unknown function with special focus in transcriptional regulators. The authors invented a gene neighborhood examination tool called “Function Discovery V1.0”. This software identifies co-locations of genes within genomic neighborhoods across many organisms, associates them with their respective metabolic pathways, and by doing so, connects the pathways with their respective transcriptional regulators (TR). The software rapidly identifies and compares conserved genomic neighborhoods encoding for metabolic pathway enzymes across many organisms and identifies candidate pathways in which the unknown function proteins (TRs) may participate.

The authors surpassed the theoretical prediction boundaries by creating a systematic approach that allowed them to test the discovery predictions and fully characterize the biological function of TRs of unknown function (effector and DNA operator). The approach tested metabolites within the predicted metabolic pathway for binding. The team used a mass spectrometry-based platform, screened for the DNA operator using microarrays, and confirmed the triad (Effector/TR/DNA operator) regulatory activity using electrophoretic and fluorescence techniques. The creation of a systems biology approach for the discovery, prediction, and validation of unknown protein function is a significant leap in our ability to understand living organisms. This approach will help close the gap between gene sequence and protein function – allowing scientists to begin deriving phenotype from genotype.

Reference: “Automated Genomic Context Analysis and Experimental Validation Platform for Discovery of Prokaryote Transcriptional Regulator Functions,” BMC Genomics15, 1142 (2014); doi:10.1186/1471-2164-15-1142. http://www.biomedcentral.com/1471-2164/15/1142 Authors include: Ricardo Martí-Arbona, Kristy L. Nowak-Lovato, Melinda S. Wren, Pat J. Unkefer and Clifford J. Unkefer (Bioscience Division); and Fangping Mu (Theoretical Division).

Laboratory Directed Research and Development (LDRD) funded the work, which supports the Global Security mission area and the Science of Signatures science pillar through insight into the link between gene sequence and protein function. Technical contact: Ricardo Martí-Arbona