MOLECULAR BIOLOGY AND EUKARYOTIC GENE REGULATION
Learning outcomes of the course unit
The aim is to provide an in-depth knowledge on the mechanisms controlling eukaryotic gene expression at both the transcriptional and post-transcriptional level, taking into account the information recently made available by genomics. This will be pursued through a unifying conceptual framework pointing to general regulatory strategies shared by prokaryotes and eukaryotes and by different phases of the genetic information transfer process (transcription, mRNA splicing and other post-transcriptional events, controlled modification/degradation and subcellular localization of proteins, signal transduction to the cell nucleus). Both theoretical and practical aspects of gene expression control will be considered, with special emphasis on post-genomic technologies such as “transcriptomics” and “phenomics”. The application of this kind of studies to “Drug Discovery” will also be discussed.
Course contents summary
Structure and functionality of the eukaryotic genome: repeated sequences and gene duplication, transposable elements, simple and complex transcription units, multigene families, regulatory sequences and differential expression of paralogous genes, duplication, modification and divergent evolution of genes coding for regulatory proteins.
Chimeric gene constructs and the “reporter gene” approach: homologous promoter/heterologous coding sequence; heterologous promoter/heterologous regulatory protein/heterologous coding sequence (“reporter gene”).
Regulatory strategies relying upon “regulated recruitment”; activator bypass experiments and the “two-hybrid” technology; squelching and transcription factor decoys; regulated recruitment and cooperativity; other types of gene regulation processes (RNA polimerase modification; DNA modification; RNA mediated repression).
Eukaryotic RNA polymerases, transcriptional regulators and promoters. General transcription factors (GTFs); “mediator”, chromatin remodelling and histone modification (HAT, HDAC) components; pre-initiation complex formation; post-initiation events; the Saccharomyces cerevisiae activator Gal4; the negative regulators Gal80 and Mig1.
Chromatin and nucleosomes; regulatory role(s) of histone tail modification (acetylation/deacetylation); chromatin immunoprecipitation (CHIP); the other eukaryotic transcription systems (RNA pol I and RNA pol III); combinatorial control: mating type regulation in Saccharomyces cerevisiae; telomers and their associated regulatory effects; insulator sequences and other higher-order control elements.
Enhancers and signal integration; different modes of control of transcriptional regulators; nuclear receptors; SREBP; Tubby; Notch and APP; NF-kB, TAT/TAR, Rb and cyclins; RNA as a transcriptional regulator: riboswitches, miRNAs, siRNAs and RNA interference; systematic gene disruption, knockout mutant arrays, “phenomic” analysis and its utilization for the identification of novel drug targets and the development of new validation systems.
Other cellular processes relying on “regulated recruitment”: ubiquitin labelling and controlled degradation of selected target proteins, mRNA splicing and its regulation, hormone-receptor interaction and signal transduction pathways.
Watson J.D., Backer T.A., Bell S. P., Gann A., Levine M., Losick R.
MOLECULAR BIOLOGY OF THE GENE (Fifth Edition)
CSHL Press; Pearson/Benjamin Cummings Publishers, 2004
The course, which is made up by lectures and exercises, is flanked by a practical laboratory tutorial dealing with host/vector systems and the use of recombinant technologies for the production of heterologous proteins.
Assessment methods and criteria