Learning objectives
KNOWLEDGE AND UNDERSTANDING
The main goal of the course is the understanding of the fundamental biochemical aspects of nucleic acids. The first part focusses on the DNA structural features that are the key aspects of stability, informational content and expression of the genetic material. The molecular mechanisms of DNA replication, DNA repair, transcription and translation are analysed in depth. A large part of the course is dedicated to the understanding of fundamental regulatory strategies operating in bacteria and bacteriophages and their possible implications for the control of gene expression in more complex organisms.
APPLYING KNOWLEDGE AND UNDERSTANDING
The educational objective of the course is to attain the skills necessary for a critical analysis of the molecular and biochemical mechanisms of life, as well as for the understanding of the basic elements of the main cellular processes.
MAKING JUDGEMENTS
The course is aimed at increasing the ability to critically analyze the molecular mechanisms of life.
COMMUNICATION SKILLS
The course includes significant activity of classroom discussion aimed at developing the ability of students to trasmit the acquired competence in support of their arguments.
LEARNING SKILLS
The many advancements in scientific research, particularly in the field of molecular biology require a continuous updating of skills. For this reason, the course aims to provide the necessary tools to achieve a wider knowledge and to align skills to the advancement in biological research.
Prerequisites
For a better understanding of the course it is essential for the student to know the basic principles of chemistry, biochemistry and genetics.
Course unit content
STRUCTURE OF NUCLEIC ACIDS
DNA REPLICATION
DNA REPAIR
TRANSCRIPTION
RNA SPLICING
TRANSLATION
GENE REGULATION IN PROKARYOTES
MOLECULAR BIOLOGY TECHNIQUES
Full programme
STRUCTURE OF NUCLEIC ACIDS
Properties of genetic material: The transforming principle, Avery's discovery, Chargaff rule; chemico-physical properties of nucleotides; chemical modifications, protonation and keto-enolic tautomerism of nitrogenous bases; the primary structure of DNA; the thermodynamics of the phosphodiester bond; weak bonds and strong bonds; the double helix and base pairing according to the Watson and Crick model; semi-conservative DNA replication; alternative secondary DNA structures (DNA A, DNA Z, DNA H); repeated direct, inverted and specular sequences; and intrinsic curvature of DNA; structural DNA recognition elements (read-out); specific DNA-protein interactions; stability of the double helix: fusion and renaturation of DNA; primary and secondary structure and distinguishing characteristics of RNA; alkaline hydrolysis of RNA and mechanism of action of RNase A; topology elements: supercoiling, bond number and conformational variations of DNA; topoisomerase I and II; condensation of nucleic acids: histones, nucleosome, fibres and higher-order chromatin structures.
DNA REPLICATION
General outline of replication: DNA thermodynamics and synthesis mechanism; structure of the active DNA polymerase site; processivity and sliding clamp; DNA polymerase proof-reading activities; semi-discontinuous DNA synthesis: leading strand, lagging strand, Okazaki fragments and removal of primers; mechanism of action of DNA ligase; origins of replication; DNA polymerase III, replisome structure and assembly; DNA primase, DNA helicase, DNA topoisomerase and other proteins involved in replication; bidirectional replication of the E. coli genome; replication of eukaryotic genomes; the problem of DNA ends.
DNA REPAIR
Point mutations; hydrolytic damage and chemical modifications of nitrogenous bases; Ames test; mismatch repair; repair by photoreactivation; repair by excision of bases; repair by nucleotide excision; repair by homologous recombination; translesion DNA synthesis; induction of SOS response.
TRANSCRIPTION
General structure of genes and prokaryotic operons; bacterial promotors: regions -10 and -35, UP elements, extended element -10; bacterial RNA polymerase structure; the sigma factor; general outline of the transcription process: initiation, elongation, termination. Transcription in eukaryotes: promotors, the pre-initiation complex, the mediator, RNA polymerase II; capping and polyadenylation of RNA; RNA polymerases I and III.
RNA SPLICING
Introns, exons and post-transcriptional processing of the primary transcripts; splicing chemistry; the spliceosome; splicing of group I and II introns; alternative splicing; RNA editing; transport of mRNA out of the nucleus.
TRANSLATION
The genetic code; messenger RNA (mRNA) structure; transfer RNA (tRNA) structure; attachment of amino acids to tRNA; aminoacyl-tRNA synthetases; the ribosome; peptide bond formation; molecular mechanism and functional phases of translation: initiation, elongation, termination; translation fidelity and energetics; the problem of broken RNAs.
GENE REGULATION IN PROKARYOTES
General principles of transcriptional regulation; positive and negative regulation of transcription; remote action; lactose operon: LacI, Cap; alternative sigma factors; NtrC, MerR and AraC; tryptophan operon (TrpR) and attenuation; transcriptional regulation and the lysis-lysogeny decision of lambda phage; structure a function of cI and cro repressors; cooperativity in the repressor bond; positive and negative transcriptional control, antitermination, antisense regulation. Lambda integration and excision by site-specific recombination.
MOLECULAR BIOLOGY TECHNIQUES
Polymerase (PCR) chain reaction; DNA sequencing by the Sanger method.
Bibliography
BIOLOGIA MOLECOLARE - Principi di funzionamento del genoma
Craig, Cohen-Fix, Green, Greider, Storz, Wolberger.
Pearson
REGOLAZIONE GENICA - autore Mark Ptashne, casa ed. Zanichelli
Understanding DNA - autore Calladine, case ed. Academic Press Terza edizione
Teaching methods
Oral lesson
Assessment methods and criteria
At the end of the course there is a written and oral examination. The written exam consists of ten questions: nine with an open answer and one with a closed answer, generally an exercise or a DNA sequence analysis. The written exam has to be completed within two hours. At each answer is assigned a score from 0 to 3 points. The sum of the scores of the ten answers represents the mark of the written exam. The oral exam consists of questions regarding unanswered parts of the written exam, plus two other theoretical questions to verify the complete preparation of the student. The oral exam can be sustained only if the mark obtained in the written exam is at least 15/30. The average of the marks obtained in the written and oral exams determines the final mark of the exam.
Other information
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