MBMB,BCHM, or CHEM 451A• This is a team taught course
– Blaine Bartholomew: 1st half– Peter Hardwicke: 2nd half
• Text is Biochemistry by Voet and VoetLehninger – 3rd edition [2004]– Others sources are Principles of Biochemistry by
Lehninger 5th edition– Supplementary material found in Genes VIII (IX)
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
COURSE OUTLINE:
First Section: Biomolecules - Bartholomew
Chapter 1: IntroductionChapter 5: Nucleic Acids, Gene Expression, and
Recombinant DNA TechnologyChapter 29: Nucleic Acid StructuresChapter 6: Techniques of Protein and Nucleic AcidsChapter 7: Covalent Structures of Proteins and Nucleic AcidsChapter 8: Three-Dimensional Structures of Proteins
Exam I Thursday September 24, 2009
Second Section: Gene Expression, Introduction to Thermodynamics
Instructor: BartholomewChapter 31: TranscriptionChapter 34: Eukaryotic Gene ExpressionChapter 30: DNA Replication, Repair, and Recombination
Instructor: HardwickeLectures 1 – 2: Outline of Principles of Thermodynamics. Lecture 3 – 4: Ligand Binding. Dissociation and association constants. ‘Affinity’. ‘Saturation’. Important non-covalent forces in ligand and substrate binding and the folding of macromolecular. Simple ligand binding. Cooperative ligand binding. Agonists and competitive antagonists.
Exam II Thursday October 29, 2009
Third Section: Protein Structure/Function and Bioenergtics
Instructor: Hardwicke
Lectures 5 - 6. Water. Autoprotonation of water. pH. Strong acids, strong bases, weak acids, weak bases. Henderson-Hasselbach equation. Effect of H2O and H+ on DG. Lectures 7-8. Amino acids. Isomers. Peptides. Isoelectricpoints (pI). Proteins. Forces involved in formation of protein structure. Primary, secondary, tertiary, and quaternary structure. Common motifs. Lecture 8-10. Myoglobin and hemoglobin as examples of protein structure and ligand binding phenomena. Chapter 10 Voet and Voet.Lecture 11-12 The Carbonic Acid - Bicarbonate Buffering System as An Example of pH Issues
Exam III on Thursday December 10, 2009 (12:35-1:50 pm)
DNA Replication, Repair, and RecombinationChapter 30
Chapter 34 pages 1467-1480 and in Genes VIII pages 657-678Chromatin and TranscriptionChapter 34
Chapter 34 pages 1456-1467 and in Genes VIII pages 631-652Regulation of TranscriptionChapter 34
Chapter 31 pages 1232-1237, Chapter 34 pages1448-1456Transcription: Part A and Part BChapter 31 & 34
Chapter 34 pages 1429-1448 and in Genes VIII pages 587-594Genomic Organization
Chapter 34 pages 1422-1429, also in Genes VIII pages 571-587Packaging of DNA and NucleosomesChapter 34
Chapter 8 pages 219-2713-Dimen Structures of ProteinsChapter 8
Chapter 6 pages 127-151, Chapter 7 pages 161-175 and pages 203-207 Methods for Working with ProteinsChapters 6-7
Chapter 5 pages 92 -122, Chapter 6 pages 145-158, Chapter 7 pages 175-203, & 207-212, Chapter 5 problems 11, 12, 13, Chapter 7 problems 12, 19
Methods for Working with Nucleic Acids Parts A and B
Chapters 5-7
Chapter 29 pages 1122-1133, Chapter 29 problems 5 & 6 DNA SupercoilingChapter 29
Chapters 1, Chapter 5 pages 80-92, Chapter 29 pages 1107-1122 Chapter 5 Problems 1, 2, 5, 6 Nucleic Acid StructureChapters 5 & 29
Suggested Readings and ProblemsLecture OutlineChapter in Voet & VoetBiochemistry
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
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
• In class participation– Report in class– List of questions or subjects given ahead of
time– worth 10 points (test will be 90 points)– Random selection
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 AssistantPayel SenOffice: Neckers Bldg., Rm. 207Phone: 453-1132Email: [email protected]
Office Hours: By appointment
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
Exo – on the opposite side of the ring as the C-5’ position
Endo – on the same side of the ring as the C-5’ position
Sugar ring pucker effects the conformation of the phosphate backbone
Sugar ring pucker effects the conformation of the phosphate backbone
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
Non-Watson-Crick base pairs
Base pairing of 9-methyladenine
Hoogsteen pairing between adenine and thymine
Base pairing between thymine and cytosine
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 atom
endo vs exo
Double Helical Structures 1. Watson Crick Structure: B-DNA
d. real DNA deviates from the ideal B-DNA formi. 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 DNA shielding 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
Questions• How do DNA or RNA modifications affect their
structure in the following examples?– Methylation of the C5 position of deoxcytidine in DNA– 4-S-deoxythymidine– Inosine
• What are in vivo examples of non Watson-Crick base pairing?
• What would a protein be recognizing if it binds to DNA independent of the DNA sequence?
• What would a protein be recognizing if it binds to DNA in a DNA sequence dependent manner?
