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
Classroom Lessons
Assessment methods and criteria
The knowledge acquired during the Molecular Biology course will be verified through a written test and an oral exam held in person. The written test, to be carried out in a maximum time of two hours, consists of ten questions aimed at assessing the degree of learning and critical analysis of the topics covered during the course. Each answer is evaluated with a score from 0 to 3 points. The oral exam involves the revision of the written test plus other questions aimed at assessing the ability to apply knowledge and understanding, as well as the autonomy of judgment and the ability to communicate ideas and concepts with clarity and properties of language. The final grade will result from a balanced evaluation of the various aspects mentioned. Honors will be awarded to students who show excellent knowledge and understanding of the subject, excellent ability to apply them, good judgment autonomy and mastery of the disciplinary vocabulary.
Other information
Class schedule, exams dates, slides and other teaching resources can be found at the url: https://elly.scvsa.unipr.it
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