Learning objectives
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.
Prerequisites
For 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
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.
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
I PRINCIPI DI BIOCHIMICA DI LEHNINGER Quinta edizione - autore David Nelson e Michael Cox, casa ed. Zanichelli
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
The assessment of learning outcomes is based on a written test and an oral report in which students are asked to describe the structure and function of an assigned protein. The oral report can be sustained after passing the written test. The written test to be held in a maximum time of two hours, consists of six questions designed to assess the degree of learning and critical analysis of the topics covered.
Other information
Class schedule, exams dates, slides and other teaching resources can be found at the url:
http://scienzebiologiche.unipr.it/cgi-bin/campusnet/corsi.pl/Show?_id=0db5
