Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: engelber@cc.huji.ac.il

1. Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: engelber@cc.huji.ac.il

2. The central dogma of molecular biology

3. DNA Transcription RNA Translation Protein

4. Could proteins multiply ?

5. What do we have RNA for?

6. Same DNA content in all cells of the mulicellular organism? What is the function of DNA? Can cells function without DNA?

11. Are these all nucleotides that appear in DNA and RNA?

14. What are the cellular functions of nucleotides?

15. Some cellular functions of nucleotides Building blocks of nucleic acids. 2. Energy carrier (ATP, GTP). 3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine). 4. Regulators in signal transduction processes. 5. Second messengers in signal transduction (cAMP, cGMP). 6. Phosphate donors in phosphorylation reactions. Involved in many more pottranslational modifications. 7. Serve as structural molecules (rRNA). 8. Activators of carbohydrates for synthesis (glycogen for example).

16. Some cellular functions of deoxynucleotides Building blocks of nucleic acids (DNA).

17. Some cellular functions of deoxynucleotides Building blocks of nucleic acids (DNA). 2. Energy carrier (ATP, GTP). 3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine). 4. Regulators in signal transduction processes (GTP). 5. Second messengers in signal transduction (cAMP, cGMP). 6. Phosphate donors in phosphorylation reactions. 7. Serve as structural molecules (rRNA). 8. Activators of carbohydrates for synthesis (glycogen for example).

20. Some deviations from the averaged Watson & Crick model The pitch angle between base pairs could be 28o - 42o. Bases could propel (deviate from planarity). Damages: kinks and covalent bonding inside the helix (usually Between bases). Presence of unusual bases (in tRNA for example) allows unusual base pairing and novel structural motifs. Presence of specific sequences (stretch of purines, palindromes, sequence repeats).

28. The driving force towards synthesis is the breakdown of PPi. Phosphodiester bond

33. Mechanism of the basic synthesis reaction of nucleic acids Addition of nucleotide involves an attack by the 3’-hydroxyl group at the end of the growing RNA molecule on the a phosphate of the oncoming NTP. Two Mg2+ ions coordinated to the phosphate groups of the NTP and to three Asp residues of the  subunit of E. coli RNA polymerase (conserved in most RNA polymerasess in nature). One Mg2+ ion facilitate the attack by the 3’-hydroxyl group on the a phosphate and the other ion facilitates the displacement of pyrophosphate. The Mg2+ ions stabilize in fact the transition (intermediate) state.

34. Polymerization of nucleotides - DNA and RNA biosynthesis 1. The reaction is directional; proceeds from 5’end to 3’end. As a result the product is asymetric (5’end different than 3’end. 2. The nucleotides (of the same strand) are always linked in a phospho-di-ester bond (a covalent bond). 3. Energy is wasted in addition of each monomer. The driving force towards synthesis is degradation of pyrophosphate. 4. The precursors are always nucleotides tri-phosphates (NTPs or dNTPs). 6. The reaction is directed by a pre exist plan (a template). (No polymerase is capable of adding nucleotides randomly). May be there are some - quite important

36. Basic characteristics of DNA Pol Is not capable of de novo synthesis. Requires: A. A template (as any other polymerase). B. A primer (RNA oligo, nicked DNA, protein?) Possesses two catalytic activities: A. A 5’ to 3’ polymerase activity. B. A 3’ to 5’ exonuclease actiivty. 3. Substrates are only dNTPs.

37. How DNA Pol is regulated? Does it possess regulatory sites?

38. DNA replication is semi-conservative DNA replication is bi-directional

39. Schematic structure of E. coli replication origin (OriC) 245 bp. 3 repeats of 13 bp sequences + 4 repeats of 9 bp sequence. These elements are highly conserved in replicationorigins of bacteria.

40. Initiation step: “opening” DNA “preparing the template before any DNA synthesis occurs.

41. First key step in replication: binding of DnaA protein molecules to the four 9 bp repeats. DnaA binding requires ATP and HU Second step: binding of DnaB (hexamerix helicase). Two hexamers bind to unwind DNA at two points creating two potential replicating forks. Third step: binding of SSBs (essential for stabilizing single strand throughout the replication process) and DNA gyrase (DNA topoII) - this step allows DnaB helicase to unwind thousands of base-pairs.

42. DnaA binds cooperatively to form a core around which OriC DNA is wrapped. At the presence of ATP DnaA melts the DNA of the A-T rich 13 bp tandem repeats. DnaA molecules recruit two DnaB-DnaC complexes, one for each replication forks. (6 DnaC monomers bind the DnaB hexamer.) Gyrase must be present to relieve topological Stress - otherwise helicase cannot further catalyze unwinding. Altogether a pre-priming complex is formed: 480 kD, 6 nm radius.

43. Initiation step has prepared the template. Moving to elongation step: Priming is required. A mechanism for bi-directionality is required. Leading strand synthesis begins with The synthesis of a short primer (10-60 n) catalyzed by primase (DnaG - special RNA Pol).

44. Both strands are sybthesized by DNA Pol3. Lagging strand: A new primer is synthesized near the replication fork. Synthesis continues until the Fragment extends as far as the primer of the previous fragment.

46. Specific structural capabilities of DNA Pol 3.

47. DnaB (helicase) + DnaG (primase) form a functional unit within the replication fork, called primosome. DNA pol3 - a dimer - one set of subunits synthesize the leading strand and other set the lagging strand. Once DNA is unwound by DnaB, DnaG associates occasionally with DnaB and synthesizes a short RNA primer. A new  sliding clamp is then positioned at the primer by the clamp-loading complex of Pol 3. When a synthesis of a fragment is completed, replication halts and the core subunits of Pol 3 dissociate from their  sliding clamp and from the new fragment.

48.  subunits on DNA