CHAPTER 4, Part 1: LECTURE TOPICS: DNA and RNA - MOLECULES OF HEREDITY

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Chapter 4 Notes: Part 1 Biochemistry 461 Fall 2010 CHAPTER 4, Part 1: LECTURE TOPICS: DNA and RNA - MOLECULES OF HEREDITY 1) DNA/RNA structures, nomenclature, shorthand conventions 2) DNA and RNA as

1 Chapter 4 Notes: Part 1 Biochemistry 461 Fall 2010 CHAPTER 4, Part 1: LECTURE TOPICS: DNA and RNA - MOLECULES OF HEREDITY 1) DNA/RNA structures, nomenclature, shorthand conventions 2) DNA and RNA as genetic material 3) General properties of DNA Double Helix 4) Basic mechanism of DNA replication 5) Important physical-chemical properties of DNA Page 1 of 31 Reminder from Chapter 25 Lectures: Nucleic acids (DNA, RNA): are polymers consisting of pentose sugars, phosphate, and bases

2 Page 2 of 31 RNA and DNA are chains of ribo (or dexyribo-)nucleosides connected by 5' to 3' phosphodiester bonds. They are polynucleotides. ** The chains have polarity, and are always written and read from 5' to 3' (left to right) as in the examples of DNA and RNA trinucleotides. (Fig.5.3)

3 Nucleic Acid Shorthand Notations: Trinucleotide shorthand example (Fig.5.7): pacg (see also Powerpoint version) Page 3 of 31

4 Page 4 of 31 DNA and RNA as genetic material: Proofs! Transformation with pure DNA (Pneumococcus, 1928, 1944)! Bacteriophage T2 DNA not Protein (1952)

5 BACTERIOPHAGE VIRUS LIFE CYCLE Scenario: attach to cell inject DNA reproduce (new DNA and proteins) kill cells release progeny (new) virus infect more cells new

6 Page 5 of 31! TMV - RNA as genetic information(1955)! Retroviruses: RNA to DNA (1970's) (Fig.5.23)

7 Page 6 of 31! PRIONS - Proteins (not DNA or RNA are transmissible agents (1970's to present)

$diffraction patterns of B-DNA in 1938.$

8 DNA (chromosomes) are long as seen by electron microsocopy of E. coli DNA (Fig.5.8) Page 7 of 31 DNA molecules are doble-helices - deduced from x-ray diffraction patterns of B-DNA in The data showed that there had to be regularly spaced units at 3.4 Angstroms along the helix. (Fig.5.10)

9 Page 8 of 31 DNA Structure: Watson-Crick Double Helix (1953)! B-DNA was the form upon which Watson and Crick derived their model. DNA occurs primarily in this form in vivo. The important features of this model of DNA are:! Two helical polynucleotide chains coil around a common axis. The chains are antiparallel in polarity.! The purine and pyrimidine bases are inside the helix, whereas the phosphate and deoxyribose units are on the outside.! The planes of the bases are perpendicular to the helix axis.! The planes of the sugars are nearly at right angles to those of the bases.! The helix diameter is 20Å.! Adjacent bases are spaced 3.4Å along the helix axis. Adjacent bases are related by a rotation of 36 degrees. Hence, the helical structure repeats after each ten residues on each chain (360 degrees, and at intervals of 34Å).! The two chains are held together by hydrogen bonds between pairs of bases. Adenine always pairs with thymine. Guanine always pairs with cytosine. [The specificity of the pairing of bases is the most important aspect of the DNA double helix. Watson and Crick deduced - while building the model - that adenine must pair with thymine, and guanine with cytosine, because of steric and hydrogen-bonding features. Chemical analysis of DNA (Chargaff, 1950) showed that A=T and G=C, as also predicted by the Watson-Crick structure.]! Any sequence of bases may occur along a polynucleotide chain. The precise sequence of bases carries the genetic information.

10 Page 9 of 31 DNA helix - flat version shows polarity and base pairing: DNA Helical ladder as In real B-DNA

11 Page 10 of 31 Space-filling version of Watson and Crick B-DNA model: (Fig.5.11a) Top View, (Figs.5.1.b and 5.13) looking down helical axis:

12 Page 11 of 31 A-T and G-C base pairs: (Fig 5.12) A-T base pair G-C base pair

13 Page 12 of 31 A three base pair stack of DNA showing:! antiparallel polarity (3'-5' and 5'-3')! bases parallel to each other and perpendicular to helix axis! dexyribose perpendicular to bases! phosphates on outside of helix

Page 13 of 31 REPLICATION OF DNA: Watson and Crick deduced that the complementary chains of a double helix are templates for each other in replication.

4-13, Fig 14, 15 ].! DNA strands with 15 N and 14 N containing bases were analyzed through several generations of E. coli cell division (and DNA replication).

14 Page 13 of 31 REPLICATION OF DNA: Watson and Crick deduced that the complementary chains of a double helix are templates for each other in replication. The base sequence of each chain determines that of daughter DNA molecules.! DNA replication is semiconservative: Shown by the Meselson-Stahl (1958) experiment [Figs. 4-13, Fig 14, 15 ].! DNA strands with 15 N and 14 N containing bases were analyzed through several generations of E. coli cell division (and DNA replication).! Changes in buoyant density (see physicochemical properties of DNA below) of old ( 15 N) vs new ( 14 N)-DNA after each generation proved that parent DNA strands were conserved during replication.

15 Page 14 of 31 DNA Replication: Primer - Template relationships Primer (free 3'-OH) (DNA) n+1 (DNA) n+2 Template (base-paired to primer) DNA Replication by DNA polymerase requires:! All four dntps (datp, dgtp, dctp, and dttp)!! Mg ++! A primer chain with a free 3'-OH end! A template strand to which the primer is base-paired! Double-stranded DNA that is fully intact and lacking a free 3'-OH end will not be replicated (ex: intact circular DNA) [***HINT: Draw your own Template-Primer complex to see how it works]

16 DNA Replication: Phosphodiester bond formation by DNA polymerase Page 15 of 31 Summary of the basic mechanism of DNA replication:! Replication is semiconservative! DNA polymerase requires a template-primer complex! dntps are the substrates for DNA synthesis! PPi breakdown to 2 moles of Pi (catalyzed by pyrophosphatase) drives DNA synthesis reaction

Page 16 of 31 Some important physical-chemical properties of DNA:! Reversible separation-reassociation of DNA strands [Denature/renature/melting temperature (T m ) proportional to %G-C bp]!

17 Page 16 of 31 Some important physical-chemical properties of DNA:! Reversible separation-reassociation of DNA strands [Denature/renature/melting temperature (T m ) proportional to %G-C bp]! Buoyant density analysis of DNA shows it is proportional to %G-C bp.! Enormous range of lengths - m to cm lengths! Conformation of DNA can be linear, circles (open/supercoiled), Single- (SS) or double-stranded (DS). Reversible strand separation of DNA by heat (and other denaturants)

18 Page 17 of 31! DNA UV-light absorbance by single and double-stranded DNA! Denaturation - A-T vs G-C base pairs! Melting temperature of DNA proportional to %G-C base pairs

Page 18 of 31 Enormous size ranges of DNA molecules in nature:! From about 2 microns (virus DNA) to 2.1cm (Drosophila largest chromosome) to 1.6-8.2 cm (human chromosome)!

[These DNA molecules replicate in host cells in a double-stranded replicative form (RF), which gives rise to new single-stranded viral DNA that is packaged into new virus particles].

19 Page 18 of 31 Enormous size ranges of DNA molecules in nature:! From about 2 microns (virus DNA) to 2.1cm (Drosophila largest chromosome) to cm (human chromosome)! Note: Useful conversion factors: [1 kb DNA = 10 3 base pairs = 0.34 x 10-6 meters]! Most have double-stranded DNA, but some viruses (X174) have single stranded DNA in the virus. [These DNA molecules replicate in host cells in a double-stranded replicative form (RF), which gives rise to new single-stranded viral DNA that is packaged into new virus particles].! Some DNAs are circular (E. coli chromosome, mitochondrial DNA [Fig.5.18], plasmids), some are linear (T7 DNA, lambda phage DNA)! Circular DNA molecules can be supercoiled or relaxed. Supercoiling is necessary for "packaging" in cells. [Fig.5.18] Definitions: 1 kb (kilobase) of DNA or RNA = 1,000 bases long (single or double-stranded). 1 kb double-stranded DNA = 0.34 m long 1 kb 1 kb double-stranded DNA = 660 kd

20 Page 19 of 31 SUMMARY: DNA and RNA - Molecules of Heredity 1) DNA/RNA structures! Know nomenclature + shorthand conventions 2) DNA and RNA as genetic material! Transformation with DNA! T2 DNA not Protein! TMV RNA! RNA to DNA (Retroviruses)! PRIONS (Proteins as transmissible agent?) 3) General properties of Watson-Crick DNA Double Helix! Antiparallel strands! Right-handed helix! 10 bp/helix turn; 3.4Angstroms/bp! bases on inside and parallel! bases perpendicular to deoxyribose-p chain! A-(double bond)-t and G-(triple bond)-c base pairs 4) Basic mechanism of DNA replication! Semiconservative! DNA polymerase I (template-primer complex)! PPi --> 2Pi (pyrophosphatase) drives DNA synthesis 5) Some important physical-chemical properties of DNA! Buoyant density (proportional to %G-C)! Denature-renature - melting (T, proportional to %G-C)! m to cm lengths! linear, circles (open/supercoiled), SS, DS! RNA molecules usually exist as partially double-stranded structures

Molecular Biology I. The Chemical Nature of DNA. Dr. Obaidur rahman

Molecular Biology I. The Chemical Nature of DNA. Dr. Obaidur rahman Molecular Biology I The Chemical Nature of DNA Dr. Obaidur rahman Characteristics of Genetic Material The coding instructions of all living organisms are written in the same genetic language that of nucleic