Learning objectives
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".
ACQUIRING KNOWLEDGE AND UNDERSTANDING.
Students are expected to acquire a detailed knowledge at the molecular
level of some key cellular processes (mainly related to gene expression control at the transcriptional, post-transcriptional and translational level), besides familiarity with the main
molecular methodologies underlying the experimental discoveries they will be learning about.
APPLYING KNOWLEDGE AND UNDERSTANDING.
Through the guided analysis of the key experiments and structures that led to our current understanding of the main processes involved in the management and transfer of biological information, students will gain the ability (i.e., competecence and criticisim) to addreess the experimental study of novel (or unrelated) bio-molecular processes and to plan meaningful (i.e., carefully controlled) experiments in the field of molecular biology
Prerequisites
For optimal understanding of the course, students should have a good basic knowledge of General and Organic Chemistry, and Physics.
Course unit content
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.
Full programme
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
NGS and genome editing technologies
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
posttranscriptional 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 posttranscriptional regulation
Bibliography
Watson J.D., Backer T.A., Bell S. P., Gann A., Levine M., Losick R. BIOLOGIA MOLECOLARE DEL GENE Zanichelli, VI edizione
Amaldi, Benedetti, Pesole, Plevani
BIOLOGIA MOLECOLARE
Ed. Ambrosiana.
Ptashne, M. REGOLAZIONE GENICA, Zanichelli (2006)
Teaching methods
Classroom lectures making extensive use of images, schemes and real experimental data. The course is flanked by exercises, test questions and practical laboratory tutorials.
Assessment methods and criteria
Evaluation of the expected achievements will be based on a written test comprising 10 questions. At least one question will bear on calculations and quantitative analyses typical of a Molecular Biology laboratory. Other questions (including multiple choice questions) will bear on specific issues dealt with during the course. A final question will deal with the description (including practical schemes) of a Molecular Biology technique, with examples on the results to be expected in a specific context of application.
The test is designed to allow a detailed evaluation of the theoretical and practical knowledge on the various molecular processes illustrated during the course as well as the ability to apply such knowledge to address and solve specific experimental problems.
Other information
2030 agenda goals for sustainable development
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