MOLECULAR BIOLOGY AND INTEGRATED BIOTECHNOLOGIES LABORATORY III
Learning outcomes of the course unit
The main goal of this course is to provide the necessary background for understanding the key aspects of nucleic acid structure, thus explaining the peculiar stability, informational content and 'readability' properties of DNA and RNA. Special attention will be given to the interaction between nucleic acids and regulatory proteins and to various forms of reciprocal adaptation aimed at maximizing the specificity and regulatory potential of such interactions. Specific case studies deal with bacterial proteins involved in DNA replication and repair, restriction/modification enzymes, RNA polymerase and other transcriptional proteins, ribosomes and other key components of the translational machinery. The different regulatory strategies utilized by bacteria and phages, and their implications (conceptual similarities) with respect to the gene expression control mechanisms operating in more complex organisms will also be examined.
Another general goal is to provide conceptual proof, through theoretical and practical examples (e.g., DNA polymerase, DNA ligase, restriction/modification enzymes, PCR, enzymatic DNA sequencing, host/vector systems and their different types of regulation), of the close connection between basic Molecular Biology and its many applications in the field of "recombinant DNA technology".
Course contents summary
The "gene" and some basic features of genes and the informational flux in prokaryotes and eukaryotes; chemical and biological properties of nucleic acids; DNA structure; the double helix and alternative secondary structures of DNA; B-DNA stability; distinctive features of RNA; tertiary structure and compaction of nucleic acids; basic principles of DNA topology. constitute
DNA replication and modification: the basic scheme of replication; DNA polymerase I and DNA ligase: key components of the replication system and important tools of recombinant DNA technology; DNA polymerase III and "replisome" assembly; replication fidelity; specialized replication systems (DNA methylation and bidirectional replication of the Escherichia coli genome; plasmid DNA; phage genomes); linear replicons and the problem of replication termination; the "polymerase chain reaction" (PCR); dideoxynucleotide-interrupted replication and enzymatic sequencing of DNA; DNA restriction and modification; an overview of chemical mutagenesis, DNA repair, recombination and transposition.
Gene transcription: the transcription process and its phases; bacterial promoters; RNA polymerase; sigma and rho factors; positive and negative regulation of transcription; transcriptional control at the level of initiation; termination and antitermination; the lactose operon (LacI, CAP), the tryptofan operon and its regulation through repression (TrpR) and attenuation; other biosynthetic operons and the SOS system; post-transcriptional modifications.
Protein synthesis: overview; the genetic code: tRNA structure and function; structure of prokaryotic mRNAs; translation factors; fidelity and energetics of translation; ribosome structure; protein synthesis regulation (autogenous control) and post-translational modification of proteins.
Phage lambda as an integrated, environmental signal responsive regulatory system; regulatory circuits controlling the lysis/lysogeny decision; positive and negative transcriptional control; anti-termination; "antisense" regulation; programmed mRNA instability and post-transcriptional regulation.
Il corso, costituito da lezioni frontali ed esercizi, è affiancato da una esperienza di laboratorio dedicata ai sistemi ospite/vettore e all'impiego delle tecnologie ricombinanti per la produzione di proteine eterologhe.
Watson J.D., Backer T.A., Bell S. P., Gann A., Levine M., Losick R. BIOLOGIA MOLECOLARE DEL GENE Zanichelli, 2005 Ptashne, M. REGOLAZIONE GENICA Zanichelli, 2006
Assessment methods and criteria