1. 1 Molecular Biology Medicine Part II b Ettore Sansavini Health Science Foundation – ONLUS Lugo (Ravenna), Italy SWITH Carlo Ventura Professor of Molecular Biology University of Bologna, Italy

2. The analysis of specific patterns of gene expression is major challenge in the understanding of complex processes including cell proliferation and differentiation, as well as tissue organization into a specific architecture. It is now increasingly becoming evident that genomic sequence represents only one level of genetic complexity and that disclosing the ordered and timely patterning of gene expression involves another level of complexity of equal magnitude in the definition and biology of living organisms Gene Expression

3. Eukaryotic RNA polymerases Type Location Cellular transcriptsEffects of  − amanitin ------------------------------------------------------------------------------------------------------- INucleolus18S, 5.8S, and 28S rRNAInsensitive IINucleoplasmmRNA precursors and snRNAStrongly inhibited IIINucleoplasmtRNA and 5S rRNAInhibited by high concentrations -------------------------------------------------------------------------------------------------------

5. Factor (Abbreviation)CompositionFunction TFIIA (IIA)2 or 3 subunitsstabilizes binding between TFIID and promoters TFIIB (IIB)single subunitinteraction between TFIID and polII-TFIIF TFIID (IID)TBP (TATA box- binding protein)binding to TATA box 8 - 10 TAF II 's (TBP-associated proteins)interaction with promoter elements and with gene-specific transcription factors TFIIF (IIF)2 subunitssomewhat like sigma in prokaryotes, this protein causes RNA pol II to bind to the complex assembly at the promoter TFIIE (IIE)2 subunitsrequired for binding and stimulation of transcription TFIIH (IIH)complex kinase activity (associated kinase activates polII by phosphorylation), helicase activity

16. A complex of CPSF (cleavage and polyadenylation specificity factor) (the blue ball), CF (cleavage factors) I and II (the brown balls) and CstF (cleavage stimulation factor) (the gray ball) bind to these sequences. A cleavage occurs and CPSF remains and is joined by PAP (poly [A] polymerase) (the red ball). PAP begins the synthesis of poly [A], resulting in the addition of the first 10 A's. Finally, PAB II (pol [A] binding protein II) joins the reaction, stimulating the synthesis of poly [A], extending the tail t about 200 A residues.

20. RNA editing of apo-B pre-mRNA. The apo-B mRNA produced in the liver has the same sequence as the exons in the primary transcript. This mRNA is translated into Apo-B100, which has two functional domains: a N-terminal domain (green) that associates with lipids and a C-terminal domain (orange) that binds to LDL receptors on cell membranes. In the apo-B mRNA produced in the intestine, the CAA codon in exon 26 is edited to a UAA stop codon. As a result, intestinal cells produce Apo-B48, which corresponds to the N-terminal domain of Apo-B100. [Adapted from P. Hodges and J. Scott, 1992, Trends Biochem. Sci. 17:77.]

21. RNA editing is not confined to apolipoprotein B. Glutamate opens cation-specific channels in the vertebrate central nervous system by binding to receptors in postsynaptic membranes. RNA editing changes a single glutamine codon (CAG) in the mRNA for the glutamate receptor to the codon for arginine (read as CGG). The substitution of Arg for Gln in the receptor prevents Ca2+, but not Na+, from flowing through the channel. RNA editing is likely much more common than was previously thought. The chemical reactivity of nucleotide bases, including the susceptibility to deamination has been harnessed as an engine for generating molecular diversity at the RNA and, hence, protein levels.

22. REGULATION OF EUKARYOTIC GENE EXPRESSION TRANSCRIPTION FACTORS

25. General Transcriptional Machinery

26. Zinc Fingers

30. Zinc Fingers and DNA Bending

31. R R hsp90 RR R R

36. NHR and DNA Bending

45. 45 Reflected light-sheet microscopy (RLSM)

46. 46

47. (B) Maternal effect genes. The anterior axis is specified by the gradient of Bicoid protein (yellow through red). (C) Gap gene protein expression and overlap. The domain of Hunchback protein (orange) and the domain of Krüppel protein (green) overlap to form a region containing both transcription factors (yellow). (D) Products of the fushi tarazu pair-rule gene form seven bands across the embryo. (E) Products of the segment polarity gene engrailed, seen here at the extended germ band stage.

49. Histones H2A and H2B are yellow and red, respectively; H3 is purple, and H4 is green. The DNA helix is wound about the protein core. The histone tails extend from the core and are the sites for acetylation, which may disrupt the formation of nucleosome assemblages.

54. (a) Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) of the repressor Ume6 interacts with a specific upstream control element (URS1) of the genes it regulates. The Ume6 repression domain (RD) binds Sin3, a subunit of a multiprotein complex that includes Rpd3, a histone deacetylase. Deacetylation of histone N-terminal tails on nucleosomes in the region of the Ume6-binding site inhibits binding of general transcription factors at the TATA box, thereby repressing gene expression. (b) Activator-directed hyperacetylation of histone N-terminal tails. The DNA-binding domain of Gcn4 interacts with specific upstream-activating sequences (UAS) of the genes it regulates. The Gcn4 activation domain (AD) then interacts with a multiprotein histone acetylase complex that includes the Gcn5 catalytic subunit. Subsequent hyperacetylation of histone N-terminal tails on nucleosomes in the vicinity of the Gcn4-binding site facilitates access of the general transcription factors required for initiation. Repression and activation of some genes in higher eukaryotes occurs by similar mechanisms.

55. Structure of Histone Acetyltransferase. The amino-terminal tail of histone H3 extends into a pocket in which a lysine side chain can accept an acetyl group from acetyl CoA bound in an adjacent site.

56. Structure of a Bromodomain. This four-helix-bundle domain binds peptides containing acetyllysine. An acetylated peptide of histone H4 is bound in the structure shown.