mbmb,bchm, or chem 451a - siumed.edubbartholomew/-lectures/dna structure 07.pdf · chapter 26: rna...

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

Lecture notes and other class information is available online

Check out the followinghttp://www.siumed.edu/~bbartholomew/451A_Sec1.html

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

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

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

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

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)

And other helpsInstructor

Blaine BartholomewOffice: Neckers Bldg., Rm. 211 Phone: 453-6437 Email: bbartholomew@siumed.edu

Office Hours: Tues. & Thurs. after class at 2 p.m.

Teaching AssistantSwetansu HotaOffice: Neckers Bldg., Rm. 207Phone: 453-1132Email: swetansuh@yahoo.com

Office Hours: By appointment only

Science is question driven

How does DNA encode for all the characteristics found within

an organism?

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

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

Chemical structure and base composition

1. Numbering system of nucleic acids2. Phosphate linkages –

phosphoester bonds3. nucleotide composition4. sugar ring - pucker

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

Chemical structure and base composition

2. Phosphate linkages –phosphoester bonds

a. phosphomonoester bond b. phosphodiester bond c. phosphotriester bond

Chemical structure and base composition

3. nucleotide a. normal base compositionb. modified bases c. tautomers

Modified Bases

Modified Bases

Tautomers

Chemical structure and base composition

4. sugar ring -a. nucleotide vs. nucleoside

Nucleotide versus nucleoside

Chemical structure and base composition

4. sugar ring -a. nucleotide vs. nucleosideb. deoxyribose vs. ribose

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

Alkaline Hydrolysis of RNA

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'

Ring Pucker

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

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

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

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

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

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

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

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

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

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