Molecular Biology

A.Y. 2018/2019
Overall hours
Learning objectives
The main objective of the course is to gain a deep knowledge of the molecular structure of nucleic acids and proteins, their biological functions and the molecular methodologies used to reach these goals. In particular, the student will study the fundamental mechanisms of the DNA replication, repair and recombination processes, and the molecular processes controlling gene expression from DNA to proteins. Both classic experiments and innovative genome-wide approaches will be described to illustrate the structures and functions of the genomes.
Expected learning outcomes
Course syllabus and organization

Linea 1

Lesson period
First semester
Course syllabus
MOLECULAR BIOLOGY: LIFE SCIENCES AND BIOTECHNOLOGIES. A brief history of molecular biology from the discovery of DNA to the frontiers of current biology and the birth of biotechnology.

STRUCTURE OF NUCLEIC ACIDS: Nitrogenous bases, nucleosides and nucleotides. The phosphodiester linkage. DNA double helix: B,A and Z DNA. How the major and minor grooves are formed in the B DNA. Physical and chemical properties of DNA. Denaturation, renaturation and hybridization. Forces that stabilize DNA. The importance of the weak bonds. DNA topology: supercoiling and parameters that describe it (Linking number, Twist, Writhe and or density of the suphelix . Negative supercoiling is the most common: why? DNA topoisomerases. Catalytic mechanism of type I and II topoisomerases.
RNA structure. Ribose chemistry and RNA stability. Alkaline hydrolysis. Secondary and tertiary structures of RNA. The various functions of RNA in the cell. Ribozymes.

Outlines of protein structure and sequence-specific interactions of protein with DNA. The Helix-Turn-helix (HTH), Zinc-finger, Leucine zipper, bHLH motifs in DNA binding proteins. The interaction between proteins and RNA.

INTERACTION BETWEEN MACROMOLECULES: Assembling DNA into chromosomes: chromatin structure. The nucleosome: composition and structure. Features of the histones: primary, secondary and tertiary structure. Organization of chromatin. The remodeling of the nucleosomes and the modifications of the histones: writers, erasers and readers. The histone code. Summary on the dynamic structure of the chromatin.

Basic techniques of Molecular biology - 1
The ecombinant DNA techniques. Type II restriction enzymes: their nomenclature, classification, frequency and type of cut. How to monitor DNA fragmentation through conventional electrophoresis in agarose gel. Virtual restriction maps. Identification of specific DNA sequences by hybridization of denatured DNA fragments transferred from gel to filter (Southern blot/ hybridization).
Characteristics of plasmid cloning vectors. Cloning principles. Strategies to prevent the re-closure of a cloning vector. Clone to express a protein of interest (expression vector). Construction of genomic libraries and their use.
The polymerase chain reaction (PCR): principle of in vitro amplification of DNA. Temperature cycles. Thermostable DNA polymerases allowed automation.
Rapid DNA sequencing: the traditional enzymatic method of DNA sequencing (Sanger) and its automation. Brief note on the Next generation Sequencing (NGS) technologies.

The -omics era. Comparison by size, organization and gene density among the main sequenced genomes of prokaryotes and eukaryotes and relations with the biological complexity of organisms. Organization of the human genome: genes, intergenic regions, repeated tandem and interdisperse sequences. Satellite DNA, microsatellites, minisatellites and sequences derived from transposable elements, pseudogenes and hypotheses about their origin.

Overview of transcription. Universal properties of RNA polymerase. The transcription in prokaryotes. The bacterial RNA polymerase and the canonical bacterial promoter. The function of the factor . Parameters describing the reaction forming the closed binary complex, the stage of isomerization, the beginning of transcription and evasion from the promoter. Factors that influence the strength of a promoter.
Transcription in eukaryotes: the RNA polymerases I, II and III and their functional specialization. Promoter structures: DNA elements and trans-acting factors: the gene promoter for the rRNA precursor. Promoters recognized by RNA pol III. The structure of the Core promotor: cis-elements and general transcription factors for RNA pol II (TFIID, A, B, F, E, H). TATA box-binding protein (TBP) and its DNA binding. The assembly of the basal transcriptional machinery. Phosphorylation of RNA pol II CTD. Proximal and distal elements. The mediator. Transcriptional activators and co-activators. Influence of chromatin structure in the transcriptional control. Model of transcription initiation of protein-encoding genes.

THE CELLULAR RNAs AND THE MATURATION OF THE TRANSCRIPTS. Co-transcriptional modifications of primary transcripts produced by RNA pol II (capping, splicing and polyadenylation) and role of CTD. Structure of the Cap and its formation at 5 'of the transcript. Polyadenylation: polyadenylation signal and molecular mechanism of poly (A) tail formation at 3 'of the transcript. Coupling of transcription termination with polyadenylation.
Splicing of pre-mRNAs: characteristics of the GU-AG class of introns of pre-mRNAs. Splicing reactions from the chemical point of view. Splicing catalyzed by the spliceosome (snRNPs and proteins). The introns of group I, II and III. Self-splicing. Splicing defects can lead to diseases. Alternative splicing of pre-mRNAs. Splicing regulators: SR and hnRNP proteins. The hypotheses on the origin of the introns. Relationship between the number of introns per gene and the biological complexity. Summary on eukaryotic gene and mRNA structures: the example of the -globin encoding gene and the corresponding mRNA. The 5'-UTR and 3'-UTR of eukaryotic mRNAs.
Nucleus-cytoplasm transport of mRNA.
The maturation of other transcripts. Notes on the maturation of rRNA and tRNA precursors. The RNA interference phenomenon. Maturation of miRNAs. primary pre-RNA, pre-miRNA and miRNA and the RISC complex formation. Functions of miRNAs in the cell. Small interference RNA (siRNA) and hints to their applications in gene expression shutdown.

Basic techniques of Molecular biology - 2
Isolation of (polyA) + RNA. Synthesis of cDNA. The cDNA libraries.
Techniques for the evaluation of the expression of single genes. Northern blot, RT-PCR, overview of qReal time-RT PCR. Large-scale techniques to study the transcriptomes: microarray and RNA seq.

The machinery of translation. Ribosomes. The tRNAs: splicing of the tRNA precursors. Structure of the tRNA. The aminoacyl-tRNA synthetases. The mechanism of translation into prokaryotes as a paradigm. Translation initiation into eukaryotes. G protein and their role in the various phases of translation. Energy cost of protein synthesis.
Example of a negative feed-back translation control in prokaryotes: ribosomal proteins. General translational control in eukaryotes: the eIF4BP and eIF2 proteins and their regulation by phosphorylation. Adjustment of the translation of single mRNAs: the case of uORF in yeast GCN4 mRNA, control of ferritin mRNA translation: role of 5'-UTR and 3'-UTR on the translatability and stability of an mRNA. Modulation by miRNAs.

POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS: The repertoire of post-translational modifications increases the diversity of the proteome. Sorting and localization of proteins. Nuclear-cytoplasm transport: the Ran protein. The co-translational translocation at the ER level. The secretory pathway. Post-translational modifications: N-glycosylation, phosphorylation, acetylation. The protein kinases and phosphatases. The importance of protein glycosylation in the production of glycoproteins of therapeutic interest.

Universal properties of DNA polymerases. Structure, domains and catalytic mechanism. The thermodynamics of the polymerization reaction. How selectivity is achieved: the kinetic control. Proof-reading activity. Replicative and specialized DNA polymerases. The enzymology of DNA replication in prokaryotes and eukaryotes: the replisome. DNA replication initiation. The replicon-initiator model. Isolation of replicators by genetic approaches and their features. The initiation of DNA replication in bacteria and yeast. Replication termination. Telomeres. Telomerases and their function.

The various types of DNA damage. Main mechanisms of DNA repair: direct repair, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER). The mechanism of MMR and NER in E. coli. Mechanism of DNA damage tolerance. The translesion DNA synthesis (TLS) carried out by specialized polymerases. Features of the TLS DNA polymerases. Repair of the double strand break (DSB) by non-homologous end joining (NHEJ) or homologous recombination.
BIO/11 - MOLECULAR BIOLOGY - University credits: 9
Lessons: 72 hours
Professor: Popolo Laura Maria

Linea 2

Lesson period
First semester
Course syllabus
Brief introduction on protein structure
The molecular features of genes
The chemical nature of the genetic material: the discovery of the DNA
DNA structures
Chemical-physical properties of DNA
RNA structures: chemical-physical properties of RNA
DNA topology and DNA topoisomerases

Brief introduction on gene functions
Introduction on the control of gene expression
Short summary on the flow of genetic information and the discovery of the genetic code
The different levels of the gene expression

Organization and evolution of genes, chromosomes and genomes
The structure and plasticity of prokaryotic genomes.
Organization of eukaryotic genomes. The structure of eukaryotic chromatin. Gene families, DNA repeats, satellite DNA, microsatellites, minisatellites, transposones and retrotransposones. The DNA test.

Methods to study nucleic acid and proteins structure as well as gene expression
Spectrophotometric and ultracentrifugation techniques
DNA denaturation and renaturation: probes and hybridization.
DNA cloning techniques: The recombinant DNA technologies. Types of cloning vectors.
The polymerase chain reaction (PCR) and its multiple applications.
DNA and cDNA libraries and their uses.
Electrophoresis and other techniques to separate nucleic acid and proteins: Southern, Northern, and Western blotting.
Classic method for DNA sequencing and the development of next generation sequences procedures.
Methods to study gene expression and mutagenesis.
Knock out and other methods to switch off gene expression. Various genome editing methods.
Genetic, biochemical and molecular biology approaches to study protein-DNA and protein-protein interactions.
Experimental approaches for genome and proteome wide analyses.
The development of Bioinformatics tools.
The relevance of model organisms to study genomes and their expression.

DNA replication
DNA replication models
Gene and factors involved in DNA replication in prokaryotes, eukaryotes, DNA and RNA virus .
Control of DNA replication.

DNA repair
Different types of DNA damages.
Different types of DNA repair mechanisms.
The control of the genome integrity and the DNA damage signal transduction pathway.
Genome integrity checkpoints and cancerogenesis.

DNA recombination
Meiotic recombination
General and site-specific recombination
Recombination models
DNA transposition
Molecular mechanisms and factors involved in DNA recombination

DNA transcription
Prokaryotes as paradigms for the control of gene expression at the transcriptional level.
The eukaryotic RNA polymerases and their promoters.
General transcription factors in eukaryotes.
Transcriptional activators and repressors in eukaryotes
Impact of the chromatin structure on the control of transcription.
Processing of the RNA transcripts
The splicing process
The control of the RNA transcripts stability
Regulation of gene expression through RNA splicing and transcript stability

RNA translation (protein synthesis)
Mechanism of RNA translation
RNA translation and the control of gene expression
Post-translational regulation of gene expression
Protein stability
Post-translational controls (phosphorylation, ubiquitination and other types of protein modifications)
BIO/11 - MOLECULAR BIOLOGY - University credits: 9
Lessons: 72 hours
Professor: Pellicioli Achille
Wednesday 10.30-12.30 and Friday 10.30-11.30
Monday and Friday 11.30-13.30
office room at the 5th floor B