Welcome Each of You to My Molecular Biology Class

Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/08/05

Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

Maintenance of the Genome Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/08/05

CHAPTER 6 The Structures of DNA and RNA How do the structures of DNA and RNA account for their functions?

OUTLINE 1.DNA StructureDNA Structure 2.DNA TopologyDNA Topology 3.RNA StructureRNA Structure

DNA STRUCTURE

DNA STRUCTURE (1) DNA is composed of polynucleotide chains Structure: twisting around each other in the form of a double helix.

Schematic model Space-filling model

Nucleoside & Nucleotide, the fundamental building block of DNA glycosidic bond phosphoester bond

DNA polarity: is defined by the asymmetry of the nucleotides and the way they are joined. Phosphodiester linkages: repeating, sugar-phosphate backbone of the polynucleotide chain

Bases in DNA purines pyrimidines adenine guanine cytosine thymine N9 N1

DNA STRUCTURE (2) Each bases has its preferred tautomeric form (Related to Ch 9)

The two strands of the double helix are held together by base pairing in an antiparallel orientation, Which is a stereochemical consequence of the way that adenine and thymine,and guanine and cytosine, pair with each other. (Related to replication and transcription) DNA STRUCTURE (3)

The Two Chains of the Double Helix Have Complementary Sequences Example: If sequence 5’-ATGTC-3’ on one chain, the opposite chain must have the complementary sequence 3’- TACAG-5’ DNA STRUCTURE (4) Watson-Crick Base Pairing (Related to replication and transcription)

The strictness of the rules for “Waston-Crick” pairing derives from the complementarity both of shape and of hydrogen bonding properties between adenine and thymine and between guanine and cytosine.

A:C incompatibility

Hydrogen Bonding Is Important for the Specificity of Base Pairing DNA STRUCTURE (5)  The hydrogen bonds between complementary bases contribute to the thermodynamic stability of the helix (why?) and the specificity of base pairing  Stacking interactions between bases significantly contribute to the stability of DNA double helix

The double helix has Minor and Major grooves (What & Why) DNA STRUCTURE (5) (See the Structural Tutorial of this chapter for details) It is a simple consequence of the geometry of the base pair.

The Major groove is rich in chemical information (What are the biological relevance?) DNA STRUCTURE (6) The edges of each base pair are exposed in the major and minor grooves, creating a pattern of hydrogen bond donors and acceptors and of van der Waals surfaces that identifies the base pair.

A: H-bond acceptorsD: H-bond donors H: non-polar hydrogens M: methyl groups

The double helix exists in multiple conformations. DNA STRUCTURE (7)  The B form (10 bp/turn), which is observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions  A form (11 bp/turn), which observed under the condition of low humidity, presents in certain DNA/protein complexes. RNA double helix adopts a similar conformation.

DNA strands can separate (denature) and reassociate (anneal) DNA STRUCTURE (8) Key terms to understand 1.Denaturation 2.Hybridization 3.Annealing/renature 4.Absorbance 5.Hyperchromicity 6.Tm (melting point)

DNA TOPOLOGY

DNA TOPOLOGY (1) Structure (1): Linking number is an invariant topological property of covalently closed, circular DNA (cccDNA) Linking number is the number of times one strand have to be passed through the other strand in order for the two strands to be entirely separated from each other.

Species of cccDNA 1.Plasmid and circular bacterial chromosomes 2.Linear DNA molecules of eukaryotic chromosomes due to their extreme length, entrainment in chromatin and interaction with other cellular components (Ch 7)

Structure (2): Linking number is composed of Twist and Writhe The linking number is the sum of the twist and the writhe. Twist is the number of times one strand completely wraps around the other strand. Writhe is the number of times that the long axis of the double helical DNA crosses over itself in 3-D space. DNA TOPOLOGY (2)

Local disruption of base pairs

Function (1): DNA in cells is negatively supercoiled; nucleosomes introduces negative supercoiling in eukaryotes Negative supercoils serve as a store of free energy that aids in processes that require strand separation, such as DNA replication and transcription. Strand separation can be accomplished more easily in negatively supercoiled DNA than in relaxed DNA DNA TOPOLOGY (3)

Function (2): Topoisomerases (P ) 1.The biological importance of topoisomerase? 2.The functional difference of the two types of topoisomerases? 3.The working mechanism of topoisomerase (See the animation for detail) DNA TOPOLOGY (4)

RNA STRUCTURE

RNA STRUCTURE (1) RNA contains ribose and uracil and is usually single-stranded

Biological roles of RNA 1.RNA is the genetic material of some viruses 2.RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3.RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4.RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5.Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

Structure (1): RNA chains fold back on themselves to form local regions of double helix similar to A-form DNA RNA STRUCTURE (2) hairpin bulge loop RNA helix are the base- paired segments between short stretches of complementary sequences, which adopt one of the various stem-loop structures

Some tetraloop sequence can enhance the stability of the RNA helical structures For example, UUCG loop is unexpectedly stable due to the special base-stacking in the loop

Pseudoknots are complex structure resulted from base pairing of discontiguous RNA segments Figure 6-32 Pseudoknot.

Non-Watson-Crick G:U base pairs represent additional regular base pairing in RNA, which enriched the capacity for self-complementarity Figure 6-33 G:U base pair

The double helical structure of RNA resembles the A-form structure of DNA. The minor groove is wide and shallow, but offers little sequence-specific information. The major groove is so narrow and deep that it is not very accessible to amino acid side chains from interacting proteins. Thus RNA structure is less well suited for sequence-specific interactions with proteins

Structure (2): RNA can fold up into complex tertiary structures RNA STRUCTURE (3) RNA has enormous rotational freedom in the backbone of its non-base-paired regions Why?

Interactions in the tertiary structure  Unconventional base pairing, such as base triples, base-backbone interactions  Proteins can assist the formation of tertiary structures by large RNA molecule

The crystal structure of a 23S ribosme

Function: Some RNAs are enzymes RNA STRUCTURE (4) Ribozymes are RNA molecules that adopt complex tertiary structure and serve as biological catalysts. RNase P and self-splicing introns are ribozymes

Structure & Function: The hammerhead ribozyme cleaves RNA by formation of a 2’,3’ cyclic phophate RNA STRUCTURE (5) See animation for detail

Homework (on the CD) 1.See the animations for DNA topology, Topoisomerase, as well as Ribozyme Structure and Activity. Answering the questions in “applying your knowledge” is required. 2.Play the structural tutorial “Introduction to the DNA structure” to better understand DNA structure 3.Finish all the critical thinking exercise

Key points for Chapter 6 1.Definitions: topoisomerase, ribozyme, double helix, DNA denaturation, Tm, linking number, pseudoknot. 2.What are the structural differences between DNA and RNA? How the structural properties of DNA and RNA determine their distinct biological functions.