分子生物学基础

nullnullIn vivo DNA binding pattern of the Polycomb Txn Factor What are the genes to which it binds? How does it affect these genes?3. What determines where it Binds??null1Nitrogenous baseSugarPhosphatenull1A2. Structure of dCTP 3. Base Tautomerism Chargaff rules - A=T, G=C helical10 layer Lines Between Cross Patterns (10 Residues Per turn)Evidence for the Double Helix1. Fiber Diffraction data: -Helical geometry -3.4 A º spacing (1Aº = 10-10 m) -34 A º pitchnullNIH (not in handout)null-2’-deoxyribose2nullSugar “Pucker” ConformationsA DNAB DNA3nullPyrimidinesPurines4nullG (Keto) G (Enol) A 99.99% 0.01%Base Tautomerization5nullBase Adenine Guanine Thymine CytosineNucleoside (Deoxy)adenosine (Deoxy)guanosine (Deoxy)thymidine (Deoxy)cytidineNucleotide (d)A (mono, di-, tri) phosphate (d)G (mono, di-, tri) phosphate (d)T (mono, di-, tri) phosphate (d)C (mono, di-, tri) phosphate61’9A very useful number: 660A very useful number: 660nullRotation About the N-Glycosidic BondN3A,B DNAZ DNA (G only)7AnullPhosphodiester Backbone8nullPitch 34 ÅRise 3.4 Å Width 20 ÅMajor GrooveMinor Groove910.4 bp/turnB-DNA: A right Handed double helix Why?nullTwist 36°9null8.5 Å11.7 Å7.5 Å5.7 ÅMajor GrooveMinor Groove10null11Note to self: Discuss forces that affect helix formation Note to self: Discuss forces that affect helix formation nullC-GT-ANIHnullABZ12nullBase Displacement Determines Groove Depthdx = +3-4 ÅZ DNAMajorMinor13nullABZMiMaMaMiMiMa12nullABZZ-DNA Phosphate Backbone is Kinked14null15Question: is all B-DNA structurally identical?Question: is all B-DNA structurally identical?Implications of structural variationImplications of flexibilitynullDegrees of freedom: 7 Torsion angles and sugar conformation5’3’(Rigid)16nullStructural Variation Defined by Bases17normalfrequentneverNever (except in intercalation)CommonCommonnull5’3’3’3’3’5’5’5’Propeller Twist Maximizes Base Stacking NIHnullPropeller TwistBuckle18TextbookReal Lifenull19Naturally Occurring Variations in Roll, Slide, TwistnullPyrimidine-Purine Steps Have Little Base Stacking Step Definition: Going along one strand of DNA in 5’to 3’ direction Four Possibles: P-Y, P-P, Y-P, Y-Y19AnullPurine-Pyrimidine Steps Have Extensive Base Stacking 19BnullFor further reading on effects of sequence on structure, “Understanding DNA-The Molecule and How it Works” By Calladine and Drew Major Conclusion: DNA structure can depend on sequence In predictable, yet complicated ways. Therefore, DNA binding proteins can recognize structure, And they can be designed to bind to highly flexible DNA. DNA Topology* *Johannes’ Favorite Subject (Students’ least favorite subject) DNA Topology* *Johannes’ Favorite Subject (Students’ least favorite subject)nullDNA Unwinding Causes Topological Problems(Transcription)Unwound Parental DuplexOver- Wound region20nullMore Topological Problems21nullProperties of Topoisomerases22nullStrand Passage Model for Topo IUnwound ComplexCleavage ComplexCovalent Tyrosine-5’PStrand PassageRe- ligationL=2L=323nullTopo I Reactions24nullModel for Topo II Mechanism25nullTopo II Reactions26nullFor a good treatment of topos, see the book: “DNA replication” Arthur Kornberg and Tania Baker