Learning outcomes of the course unit
The main objective of the course is the understanding of the fundamental biochemical aspects of nucleic acids. Particular attention will be given to the understanding the DNA structure elements on the basis of which the peculiar characteristics of stability, informational content and legibility of the genetic material will be explained. The molecular mechanisms at the basis of the DNA replication, repair, recombination, transcription and translation processes will be analysed in depth. A large part of the course will be dedicated to the understanding of the fundamental regulatory strategies operating in bacteria and bacteriophages and their possible implication for the control of gene expression in more complex organisms. The course ends with an introduction to the main molecular biology techniques such as cloning, PCR and DNA sequencing.
For better understanding of the course it is essential for the student to know the basic principles of chemistry, biochemistry and genetics.
Course contents summary
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.
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.
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.
Homologous recombination: Holliday model; RecBCD, RecA, RuvAB and RuvC complex; site-specific recombination: recombination sites, insertion, deletion and inversion; serine recombinase and tyrosine recombinase; integration and excision of lambda phage into and from the E. coli genome; control of gene expression by means of site-specific recombination; resolution of multimer circular genomes by means of site-specific recombination.
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.
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
Watson J.D., Backer T.A., Bell S.P., Gann A., Levine M., Losick R.
BIOLOGIA MOLECOLARE DEL GENE (V Edizione)
Zanichelli Editore 2005
Nelson D.L, Cox M.M.
I PRINCIPI DI BIOCHIMICA DI LEHNINGER (III Edizione)
Zanichelli Editore 2002
Zanichelli Editore 2006
Calladine C.R , Drew H.R., Luisi B.F., Travers A.A.
UNDERSTANDING DNA (III Edizione)
Academic Press 2004
The course consists of classroom lectures held with a frequency of three two-hour lectures per week. Assessment is based on a written test with a time limit of two hours and consists of ten questions to assess the level of learning and critical analysis of the topics dealt with.