Lectures II-2, 3

Required Reading:

pp. 47-55; 725-746 in FOB
pp. 103-108 in MCB (5th ed.)

Suggested Questions to Answer:

Study Exercises 4,5 and Problems 2 & 4 on p. 75 of FOB
Study Exercises 1, 2, 4, 5 and Problems 2, 6, 7 on p. 771 of FOB
Problems 1-4 on p. 144 of MCB

Learning Objectives:

Outline the "Central Dogma" of Molecular Biology
Name and briefly describe three major biosynthetic/anabolic processes that are required for the maintenance and expression of genetic information, and explain how nucleic acid sequences act as templates during each. Which type(s) of nucleic acid do NOT act as templates for macromolecular synthesis during gene expression?
Describe the signficance of prior data concerning base composition (Chargaff) and X-ray fiber diffraction (Franklin) to Watson and Crick's efforts to model the structure of DNA.
Describe the key features of the DNA double helix as described by Watson and Crick, with specific reference to: helical handedness and pitch, strand polarity, relative positions of the sugar-phosphate backbone and bases, base pairing & base stacking, and major & minor grooves. Compare and contrast the double helical structure of DNA with the regular secondary structures found in proteins.
Compare and constrast the A, B, and Z conformations of DNA. Explain which of these forms predominates in DNA under physiological conditions, and which is primarily found in double-stranded RNA.
Describe several of the non-covalent forces that act to stabilize or de-stabilize the double helix, and explain why/how helix stability is affected by base composition and salt concentration/ionic strength. Describe the processes of denaturation and annealing of DNA, and explain how a the Tm of a DNA molecule can be measured experimentally. Why is annealing/hybridization important to the field of DNA cloning/biotechnology?
Explain how single stranded nucleic acids (primarily RNA) can form secondary structures. Give an example of an inverted repeat or palindromic sequence in dsDNA, and draw the secondary structure that you would expect a corresponding ssRNA molecule to form.

Lecture Outline - Overview of Nucleic acids & Molecular Biology; Nucleic acid structure

I. Significance of Nucleic acids

The "Central Dogma" of molecular biology:

Genes contain the information that specifies protein sequences

--> The flow of genetic information is from DNA to RNA to protein

Replication (DNA synthesis) ensures the faithful transmission of genetic information from generation to generation (Fig. 3-14, FOB)

At least two additional processes are required for gene expression (the functional manifestation of genetic information) (Fig. 3-15, FOB):

1) transcription (RNA synthesis)
2) translation (protein synthesis)

Nucleic acids play two kinds of role in these processes:

Informational

DNA & RNA are uniquely suited for storage & retrieval of genetic information
Both function as templates for macromolecular synthesis (DNA directs DNA & RNA synthesis, mRNA directs protein synthesis)
As templates, DNA & mRNA are “passive” (unreactive, catalytically inactive)
Information is LINEAR: sequence per se is what matters

Dynamic

rRNAs are integral parts of ribosome (e.g., catalyze peptide bond formation)
tRNAs are central to decoding of message during translation
Additional RNAs function in splicing/ RNA processing
Specific sites in DNA and mRNA regulate replication or gene expression
Structure is essential: activity depends on 3-D conformation/folding
“Protein-like” functions of nucleic acids were more prevalent in RNA world

II. Higher order structure of DNA: the Double Helix

Watson and Crick (1953) proposed a DNA structure based on:

Fundamental chemical principles

Covalent structures of nucleotides, bond rotations (Fig. 23-4, FOB), etc.
Non-covalent interactions: H-bonding, vanDer Waals,etc.

Chargaff’s rules:

for dsDNA, G/C and A/T ratios = 1, purines = pyrimidines
G+C vs. A+T content varies depending on source of DNA

X-ray fiber diffraction results (R. Franklin) (Fig. 3-8, FOB):

Cross(X)-shape indicated a helix
Outer reflection indicated a 3.4 A repeat (rise of stacked bases)
(Structure can only be solved using crystals -not fibers)

“Secondary” considerations: the genetic material must be capable of:

Faithful replication during cell division/reproduction
Coding for proteins

Key features of the double helix:

Antiparallel: 2 strands have opposite 5'-3' orientations (Fig. 3-11, FOB)
Helix is right handed (except Z DNA) (Fig. 4-3a, MCB)

Sugar-phosphate backbones trace right-handed path on outside

Base Complementarity: bases are “inside” helix and must match: G to C, A to T (Fig. 23-1, FOB)

Accounts for Chargaff’s rules
The sequence of one strand dictates the sequence of the other

For DNA sequencing projects, both strands are analyzed to reduce errors
Sequence of one strand is reported/published – other strand is implied

Each base pair is geometrically equivalent: constant C-1’ to C-1’ distance

Base stacking: along each strand, bases overlie one another
Major and minor grooves expose opposite “sides” of each base pair

Conformational variability in the double helix (Table 23-1, Figs. 3-9 & 23-2, FOB)

Most DNA approximates the “B” form proposed by Watson and Crick

10 bp/turn (so 36 degrees per bp) – DNA in solution averages ~10.4 bp/turn
Bases are ~ perpendicular to helix axis
Bases are near the center of helix
3.4 Angstroms/bp (= rise), 34 Angstroms/turn (pitch)
Major groove is wide, minor groove is narrow

A form :

For DNA, requires low water content (unlikely in cells)
Readily forms with dsRNA (Steric hindrance of 2’-OH prevents B form)
Right-handed, but less tightly wound than B form (11 bp/turn)
Helix is wider, bases are well off center & tilted, rise & pitch are smaller

Z DNA:

LEFT-HANDED
Involves special sequences: alternating purine-pyrimidine
Rarely encountered, but may be significant for certain functions

III. Secondary structure: base pairing in ssRNA (or ssDNA): (Fig. 4-8, MCB; 23-23 FOB)

Single-stranded molecules may form:

Hairpins
Stem-loops
More complex structures

Important for 3-D structure of tRNA, rRNA, ribozymes, etc

Result from “palindromic” sequences (aka inverted repeats)

Complementary sequences must be present within the same strand
The corresponding duplex (ds molecule) will have 2-fold symmetry, with the same sequence on both strands; e.g.:

5’ ..GCGAACG………CGTTCGC.. 3’

3’ ..CGCTTGC………GCAAGCG.. 5’

Palindromes may be ~perfect (hairpins) or interrupted (stem-loops)

Helical regions in RNA are usually A-form and may be imperfect due to:

Unusual (non-Watson-Crick) base pairs (e.g., Fig. 23-19, FOB)
Unmatched bases on one strand

IV. Helix stability

Non-covalent interactions determine the stability of the double helix

H-bonds mediate base-pairing: 3 bonds for G-C pairs, 2 for A-T(U)

Little if any NET contribution to stability (water competes for these)
BUT absolutely critical: what would happen if base pairs lacked H-bonds?

Base-stacking:

van Der Waals & hydrophobic forces between bases of adjacent (not paired) nucleotides
G-C pairs contribute more than A-T
Stacking (without pairing) also occurs in ssDNA/RNA

Ionic interactions:

Intrinsic repulsion between negatively-charged phosphates
Stabilized by counterions in solution: mono- and (especially) divalent cations

Helix Denaturation (melting) and Renaturation (annealing) (Fig. 23-15, FOB)

Strand separation can be effected by:

Thermal energy
Denaturing solvents (e.g., formamide)
Low salt

UV absorbance (at 260 nm) increases upon melting (Fig. 23-16, FOB)
Melting curves: (Fig. 23-17, FOB; Fig. 4-6, MCB)

Relatively sharp transition indicates cooperativity (“all or none” behavior)
T_m = temp. required for 50% denaturation (at equilibrium)
Non-cooperative curves for ssDNA/RNA reflect progressive disruption of stacking interactions

Stability (melting temperature):

Increases with G-C content
Decreases if mismatched bases are present

Renaturation:

Requires temp. below (but near) T_m (Fig. 23-18, FOB)
Basis for various hybridization methods (Southern blotting, cloning, etc)