Learning outcomes of the course unit
The main goal of this course is to provide the necessary background for understanding the key aspects of nucleic acid structure, thus explaining the peculiar stability, informational content and "readability" properties of DNA and RNA. Special attention will be given to the interaction between nucleic acids and regulatory proteins and to various forms of reciprocal adaptation aimed at maximizing the specificity and regulatory potential of such interactions. Specific case studies deal with bacterial proteins involved in DNA replication and repair, restriction/modification enzymes, RNA polymerase and other transcriptional proteins, ribosomes and other key components of the translational machinery.
A previous course of Biochemistry is strongly recommended.
Course contents summary
The ''gene'' and some basic features of genes and the informational flux in prokaryotes and eukaryotes; chemical and biological properties of nucleic acids; DNA structure; the double helix and alternative secondary structures of DNA; B-DNA stability; distinctive features of RNA; tertiary structure and compaction of nucleic acids; basic principles of DNA topology.
DNA replication and modification: the basic scheme of replication; DNA polymerase I and DNA ligase: key components of the replication system and important tools of recombinant DNA technology; DNA polymerase III and ''replisome'' assembly; replication fidelity; specialized replication systems (DNA methylation and bidirectional replication of the Escherichia coli genome; plasmid DNA; phage genomes); linear replicons and the problem of replication termination; the ''polymerase chain reaction'' (PCR); dideoxynucleotide-interrupted replication and enzymatic sequencing of DNA; DNA restriction and modification; an overview of chemical mutagenesis, DNA repair, recombination and transposition.
Gene transcription: the transcription process and its phases; bacterial promoters; RNA polymerase; sigma and rho factors; positive and negative regulation of transcription; transcriptional control at the level of initiation; termination and anti-termination; the lactose operon (LacI, CAP), the tryptofan operon and its regulation through repression (TrpR) and attenuation; other biosynthetic operons and the SOS system; post-transcriptional modifications.
Protein synthesis: an overview; the genetic code; tRNA structure and function; structure of prokaryotic mRNAs; translation factors; fidelity and energetics of translation; ribosome structure; protein synthesis regulation (autogenous control) and post-translational modification of proteins.
Phage lambda as an integrated, environmental signal responsive regulatory system; regulatory circuits controlling the lysis/lysogeny decision; positive and negative transcriptional control; anti-termination; ''antisense'' regulation; programmed mRNA instability and post-transcriptional regulation.The accompanying practical course will deal with the main molecular biological methodologies for the construction, isolation, identification by restriction/gel electrophoresis analysis and transfer into bacterial cells of recombinant plasmid DNA molecules. As a summarizing demonstration, a recombinant protein production experiment will be conducted in genetically transformed Escherichia coli cells, that will be induced to express eukaryotic (e.g., GFP) or prokaryotic (e.g., LacZ) genes coding for readily detectable proteins.
Watson J.D., Backer T.A., Bell S. P., Gann A., Levine M., Losick R.
MOLECULAR BIOLOGY OF THE GENE (Fifth Edition)
CSHL Press; Pearson/Benjamin Cummings Publishers, 2004
Voet D., Voet J.G., Pratt C.W.
FUNDAMENTALS OF BIOCHEMISTRY
John Wiley & Sons Inc., 1999
A GENETIC SWITCH (Third Edition)
CSHL Press, 2004
Additional teaching material dealing with phage lambda regulation and with some aspects of the practical course ('Expression vectors and heterologous protein production in bacterial hosts') is available to the students at http://biotecnologie.unipr.it/cgi-bin/campusnet/home.pl ("Corso di Biologia Molecolare").
The course, which is made up by lectures and exercises, is flanked by a practical laboratory tutorial dealing with host/vector systems and the use of recombinant technologies for the production of heterologous proteins. The final evaluation will rely upon a written test comprising questions and problems.