Biochem Handout #3: Nucleic Acids

1. Biochem Handout #3: Nucleic Acids

2. Both DNA and RNA are polynucleotides. Biochem Handout #3: Nucleic Acids A polynucleotide chain has a sense or directionality. The phosphodiester linkage between monomer units is between the 3’ carbon of one monomer and the 5’ carbon of the next. Thus, the two ends of a linear polynucleotide chain are distinguishable. Sequences of polynucleotides are written 5’  3’ . The sequence of bases is also known as the primary (1o) structure. RNA containsribose DNA contains 2’-deoxyribose

3. The two types of heterocyclic bases are derivatives of purine and of pyrimidine. The bases absorb uv light around 260 nm (see spectrum here)

4. Nucleoside = nitrogenous base + ribose. Nucleotide = nitrogenous base + ribose + phosphate.

5. Chem draw structures

6. Right column: How polynucleotides are actually formed. Each monomer is presented as an NTP to be added to the chain. Cleavage of the NTP provides the free energy that makes the reaction thermodynamically favorable. The enzymes catalyzing such reactions are called polymerases. Left column: Formation of a polynucleotide by a hypothetical dehydration reaction.

7. Basepairing in DNA • A-T and G-C are the base pairs in the Watson–Crick model of DNA. • Note the base pairing occurs between the keto tautomers. • The AT pair has two hydrogen bonds. • The GC pair has three hydrogen bonds. • The complementary, two-strand structure of DNA explains how the genetic material can be replicated.

8. Watson-Crick Model for the Double Helix (B-DNA) 2 antiparallel strands running in opposite directions Sugar phosphates on outside, with deoxyribose rings running parallel to the helical axis Bases on inside, perpendicular to helical axis Each base step contributes a rise of 3.4 Å and a rotation of 36o G pairs with C, A with T Base sequence carries genetic information

9. Secondary and Tertiary Structure of Nucleic Acids This view down the helix axis shows how the base pairs stack on one another, with each pair rotated 36° with respect to the next.

10. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology • A side view of the base pairs shows the 0.34-nm distance between the base pairs. • This distance is called the rise of the helix.

11. Secondary and Tertiary Structure of Nucleic Acids A space-filling model of DNA. The DNA molecule as modeled by Watson and Crick is shown here with each atom given its van der Waals radius. This model clearly shows how closely the bases are packed within the helix. Note the location of the major and minor grooves.

12. Handedness of the DNA Helix

13. Secondary and Tertiary Structure of Nucleic Acids A model for DNA replication: • Each strand acts as a template for a new, complementary strand. • When copying is complete, there will be two double-stranded daughter DNA molecules, each identical in sequence to the parent molecule.

14. Three models of DNA replication. Experimental evidence supports the semiconservative model. Brown = parental DNA Blue = new DNA

15. Secondary and Tertiary Structure of Nucleic Acids Ideal B form B-form DNA from crystal structure The two major forms of polynucleotide secondary structure are called A and B. Most DNA is B form, RNA is A form Double-stranded RNA and DNA-RNA hybrids are in the A form.

16. Secondary Structures: A form vs B form Comparison of the two major forms of DNA.

17. DNA in cells • DNA in cells can differ in size and shape. • DNA can be from thousands to millions of base pairs in length. • DNA can be circular or linear; Circular DNA can be relaxed or supercoiled • Every organism carries in each of its cells at least one copy of the total genetic information possessed by that organism. This is referred to as the genome. • The human genome consists of about 1 x 109 (one billion) bp of DNA, distributed in 23 chromosomes. Bacteriophage double-strand DNA (linear) bacterial DNA (circular)

18. Secondary and Tertiary Structure of Nucleic Acids • The DNA or RNA sequence is a primary structure, held together by covalent bonds • The regular folding patterns observed in the A- and B-DNA double helices are referred to as their secondary structures, held together by non-covalent hydrogen bonds. • The high-order folding of DNA’s secondary structure is called its tertiary structure. These structure are also held together by non-covalent interactions.

19. Secondary and Tertiary Structure of Nucleic Acids • RNA molecules are usually single-stranded. • Most have self-complementary regions that form hairpin structures. • Some have well-defined tertiary structures.

20. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology DNA replication is the copying of both strands of a duplex DNA to produce two identical DNA duplexes. The replication of DNA is accomplished by a complex of enzymes called the replisome. DNA polymerase has multiple functions. As parental DNA strands unwind, forming a replication fork, DNA polymerase guides the pairing of incoming dNTP, each with its complementary partner on the strand being copied. It then catalyzes the formation of the phosphodiester bond to link this residue to the new growing chain.

21. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology • Each of the parental DNA strands serves as a template, specifying the sequence of a daughter strand. • DNA polymerase adds nucleotides, one at a time, to the growing daughter strand, which can be considered a primerto which nucleotides are added as the daughter DNA strand grows from its 5’ end toward its 3’ end. • DNA polymerase also “proofreads” the addition before proceeding to add the next residue. • This proofreading contributes to the high overall accuracy of replication. • Because the two DNA strands run in opposite directions, one daughter strand is elongated in the same direction as that of the replication fork while the other is formed in the reverse direction.

22. Transcription • Transcription is the copying of a DNA strand into a complementary RNA molecule. • Note that U in the new RNA pairs with A in the DNA template.) • Another distinction from DNA replication is that only one of the two DNA strands, the template strand, is copied.

23. Translation • the protein-coding information is “read” in blocks of three nucleotides, or codons, each of which specifies a different amino acid. (genetic code) • Complementary copies of the genes to be expressed are transcribed from DNA in the form of messenger RNA (mRNA) molecules.

24. The Biological Functions of Nucleic Acids: A Preview of Molecular Biology • The flow of genetic information in a typical cell. • DNA can both replicate and be transcribed into RNA. • Messenger RNAs are translated into protein amino acid sequences.

25. Plasticity of Secondary and Tertiary DNA Structure • Self-complementarity in a base sequence allows a chain to fold back on itself and form a base-paired, antiparallel helix or hairpin structure • Double hairpins, often called cruciform(cross-like) structures, can be formed • in some DNA sequences. • To form this structure, the sequence must be palindromic. • The word palindrome is of literary origin and usually refers to a statement that reads the same backward and forward, such as “Able was I ere I saw Elba.” How self-complementarity dictates the tertiary structure of tRNA.

26. Plasticity of Secondary and Tertiary DNA Structure

27. Denaturation of DNA When native (double-stranded) DNA is heated above its “melting” temperature, it is denatured (separates into single strands). The two random coil strands have a higher entropy than the double helix.

28. Thermodynamics of DNA Melting At low T, DG is positive and denaturation of DNA is not favored. As T increases, • -TDS overcomes DH, making DG negative and denaturation favorable. The midpoint of the curve marks the “melting” temperature, Tm, of DNA.

29. Absorption spectra of native and denatured DNA show that native DNA absorbs less uv light than denatured DNA. This hypochromicity of double stranded DNA (dsDNA) can be used to distinguish between native and denatured forms. The change in absorbance can be used to follow the denaturation of DNA as temperature increases. An abrupt increase in absorbance, corresponding to the sudden “melting” of DNA, is seen at Tm. Denaturation (Melting) of DNA

30. Tm vs %GC Effect of base-pair composition on the denaturation temperature of DNA. The graph shows the rise in “melting” temperature of DNA as its percent (G + C) increases.

31. Manipulating DNA – Cloning Left: Creation of recombinant DNA molecules in vitro Right: Cloning a fragment of DNA into a plasmid vector and introducing the recombinant molecule into bacteria.

32. Maxam-Gilbert Sequencing