MOLECULAR BIOLOGY 2: GENE EXPRESSION REGULATION IN EUKARYOTES
Learning outcomes of the course unit
The aim is to provide in-depth knowledge of the mechanisms controlling eukaryotic gene expression at both the transcriptional and post-transcriptional level, also considering the information recently made available by genomic analysis. This objective will be attained by means of the formulation of a unifying conceptual framework revealing the existence of 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 particular reference to post-genomic technologies such as “transcriptomics” and “phenomics”. The most recent applications of these studies to “Drug Discovery” will also be discussed.
A good knowledge of basic Molecular Biology and Biochemistry is required.
Course contents summary
Structure and function 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 gene 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 based on “regulated recruitment”; activator bypass experiments and “two-hybrid” technology; squelching and transcription factor decoys; regulated recruitment and cooperativity; other types of gene regulation processes (RNA polymerase modification; DNA modification; RNA-mediated repression).
Eukaryotic RNA polymerases, transcriptional regulators and promoters. General transcription factors (GTFs); the “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; the 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 control modes in 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 use in the identification of new drug targets and the development of new validation systems; other diagnostic and therapeutic applications of microRNAs and RNA interference.
Other cellular processes based on “regulated recruitment”: ubiquitination and controlled degradation of selected target proteins, mRNA splicing and its regulation, hormone-receptor interaction and signal transduction pathways.
M. Ptashne, A. Gann, GENES AND SIGNALS, CSHL Press, 2002 (Italian edition Geni e segnali, Zanichelli, 2004)
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
H. Lodish et al., MOLECULAR CELL BIOLOGY, Freeman, 2005
R. Weaver, BIOLOGIA MOLECOLARE, McGraw Hill, 2005
Additional course material is available at http://biotecnologie.unipr.it/cgi-bin/campusnet/home.pl (Course “Molecular Biology 2: regulation of gene expression in eukaryotes”).
Lectures, including the discussion of selected scientific articles taken from the most recent literature in the field of eukaryotic gene expression. Evaluation will be performed by means of an oral test.