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MBMB,BCHM, or CHEM 451A - siumed.edubbartholomew/-lectures/DNA structure 07.pdf · Chapter 26: RNA...
Transcript of MBMB,BCHM, or CHEM 451A - siumed.edubbartholomew/-lectures/DNA structure 07.pdf · Chapter 26: RNA...
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MBMB,BCHM, or CHEM 451A• This is a team taught course
– Blaine Bartholomew: 1st half– Peter Hardwicke: 2nd half
• Text is Lehninger – Principles of Biochemistry 4th edition [2005]– Others sources are Voet and Voet 3rd edition– Supplementary material found in Genes VIII on
reserve at Morris Library
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Lecture notes and other class information is available online
Check out the followinghttp://www.siumed.edu/~bbartholomew/451A_Sec1.html
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for additional information see Genes VIII pages 657-678
Chromatin and Transcription
Chapter 28 pages 1102-1119; for additional information see Genes VIII pages 631-652Problems 3, 10, and 12
Regulation of Gene Expression in EukaryotesChapter 28
Chapter 26 pages 995-1006 in LehningerProblems
RNA MetabolismTranscription: Part A and Part B
Chapter 26
Chapter 24 pages 923-947 in Lehninger; for additional information see Genes VIII pages 571-594. Problems 5, 8, 9, 10, and 11
Genes and Chromosomes Supercoiling, Packaging of DNA, and Higher Order Structure
Chapter 24
Chapter 9 pages 306-342 in LehningerProblems 1-8, 10, 12 in Lehninger
DNA-based Information Technologies Parts A and B and Molecular Cloning
Chapters 9 & 29
Chapter 8 pages 273-305 in LehningerProblems 1, 2, 4, 5, 6, 9, 11, and 12 in Lehninger
Nucleic Acid StructureChapters 8
Suggested Readings and ProblemsLecture OutlineChapter in Lehninger
Biochemistry
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Notes and other helps
• Suggested that you print out the lecture outline before class to help you with your in-class note taking
• Don’t forget the suggested readings and problems
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COURSE OUTLINE: First Section: Nucleic Acids and Their ExpressionClick above for outline of lectures and figures Instructor: BartholomewChapter 8: Nucleotides and Nucleic AcidChapter 9: DNA-Based Information TechnologiesChapter 29: Recombinant DNA TechnologyChapter 24: Genes and ChromosomesChapter 26: RNA Metabolism Exam I September 20, 2007
Second Section: DNA and Protein Synthesis, Introduction to ThermodynamicsInstructor: BartholomewChapter 28: Regulation of Gene Expression (pages 1099-1119)Chapter 25: DNA Metabolism – only selected topics with a focus on eukaryotesProtein MethodsChapter 27: Protein Metabolism – only some selected topics
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Grading
• Each section is worth 100 points• The final exam is worth 200 points• You can drop one of the other exams for a
total number of points possible being 400 points
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First Section
• Will have a small group project• Group size of ~3 persons• Report in class and submit a written report• Project worth 10 points (test will be 90
points)• Subjects will be given out this Thursday• Presentations will start the following
Thursday (August 30, 2007)
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And other helpsInstructor
Blaine BartholomewOffice: Neckers Bldg., Rm. 211 Phone: 453-6437 Email: [email protected]
Office Hours: Tues. & Thurs. after class at 2 p.m.
Teaching AssistantSwetansu HotaOffice: Neckers Bldg., Rm. 207Phone: 453-1132Email: [email protected]
Office Hours: By appointment only
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Science is question driven
How does DNA encode for all the characteristics found within
an organism?
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Questions
• How is DNA read by the cell?• What are the distinguishing features of
DNA that accounts for its specificity?• Are there certain chemical or
mechanical properties of DNA that are vital in this process?
• What are those factors (proteins) that “read” DNA and how do they work
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Foundations
• One must first understand the structural parameters of DNA and its physical properties.
• Next it is important to know what are the variations that can be found in nature
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Chemical structure and base composition
1. Numbering system of nucleic acids2. Phosphate linkages –
phosphoester bonds3. nucleotide composition4. sugar ring - pucker
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Chemical structure and base composition
1. Numbering system of nucleic acidsa. phosphate (alpha, beta, gamma)b. base (1,2,3,...)c. sugar (1', 2', 3', ...) d. shorthand notation
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Chemical structure and base composition
2. Phosphate linkages –phosphoester bonds
a. phosphomonoester bond b. phosphodiester bond c. phosphotriester bond
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Chemical structure and base composition
3. nucleotide a. normal base compositionb. modified bases c. tautomers
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Modified Bases
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Modified Bases
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Tautomers
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Chemical structure and base composition
4. sugar ring -a. nucleotide vs. nucleoside
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Nucleotide versus nucleoside
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Chemical structure and base composition
4. sugar ring -a. nucleotide vs. nucleosideb. deoxyribose vs. ribose
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Chemical structure and base composition
4. sugar ring -a. nucleotide vs. nucleosideb. deoxyribose vs. ribosec. base-catalyzed hydrolysis of RNA
(not DNA)due to 2'-OH of RNA
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Alkaline Hydrolysis of RNA
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Chemical structure and base composition
4. sugar ring -a. nucleotide vs. nucleosideb. deoxyribose vs. ribosec. base-catalyzed hydrolysis of RNA
(not DNA)due to 2'-OH of RNA
d. ring pucker: endo vs. exo and C-3' vs C-2'
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Ring Pucker
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Double Helical Structures 1. Watson Crick Structure: B-DNA
a. antiparallel orientation, 3' vs 5' ends b. base pairing interactions
i. always a purine-pyrimidine (steric constraints)ii. tautomeric forms of bases
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Double Helical Structures 1. Watson Crick Structure: B-DNA
c. double helical parameters i. helical sense: right vs. leftii. major vs. minor grooveiii. base pairs per helical turniv. helix rise per base pair or helical pitch
distance from one step to the nextv. helical twist: angle between two adjacent base pairs
=360 deg/base pairs per turnvi. base tilt: slant of the step, not completely planarvii. glycosidic conformation: anti vs syn - figure 29-8viii. sugar ring pucker: 4 out of 5 ring atoms are nearly
planar the 5th atom is usually the C-2 or C-3 atomendo vs exo
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Double Helical Structures 1. Watson Crick Structure: B-DNA
d. real DNA deviates from the ideal B-DNA form
i. local deviations are commonii. some DNA is naturally bentiii. deviations are sequence dependent
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Double Helical Structures 2. A-DNA
a. wider and flatter than B-DNAi. very shallow minor grooveii. deeper major groove
b. tilt is 20 deg (most tilted)c. dried out DNA, 75% vs 92% humidityd. flat ribbon wound around a 6 angstrom holee. found in spores because of close packaging and
RNA-RNA/RNA-DNA hybrids assume an A-DNA like structure
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Double Helical Structures 3. Z-DNA
a. Characteristics ofi. occurs in alternating purine-pyrimidine tractsii. favored in high salt;helps eliminate
electrostatic repulsion of phosphate groups (8 vs 12 angstrom distance)
iii. methylation of deoxycytidine helps formation of Z-DNA
iv. phosphate backbone forms a zig-zagconformation
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Double Helical Structures
3. Z-DNA b. double helical parameters
i. syn vs. anti conformationpurines flip to assume syn
ii. helical sense is left handed
iii. deep minor groove, no major groove
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Double Helical Structures 3. Z-DNA
b. double helical parametersi. syn vs. anti conformation
purines flip to assume synii. helical sense is left handed iii. deep minor groove, no major groove
c. biological role of Z-DNAi. unclear at this pointii. anti Z-DNA antibody detection, artifactualiii. methylation proved to be less artificial
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Double Helical Structures 4. Unusual DNA structures
a. palindrome vs. mirror repeati. example - placement of invert repeatsii. hairpiniii. cruciform
b. Hoogsteen base pairingi. triplex formation - figure a and bii. G tetraplex - found at telomeres
c. Triple helix
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Forces that help to form the DNA double helix
1. Rigid phosphate backbone2. Stacking interactions - electronic interactions
between planar bases3. Hydrophobic interactions - highly negative phosphate
backbone vs. nonpolar bases4. Hydrogen bonding is not the most energetically
significant componentnote: maintenance of distance from the two
phosphate backbone requires Pur-Pyr5. Ionic interactions - salt stabilizes the duplex form
of DNAshielding of phosphate backbone
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Denaturation and Renaturation1. Tm: (melting temperature) temperature at which half
of the DNA is meltedMarmur-Doty equation for Tm correlated to G+C
percent and salt Tm=41.1 XG+C + 16.6 log[Na+] + 81.5
2. Denaturation is a cooperative process -caused by: heat, change in pH, organic solvents
(urea, formamide)3. Hyperchromic shift - increase of absorbance of DNA
when it goes from being double- to single- stranded40% increase in absorbance
4. Annealing:Hybridization
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