Lecture II-1,2

Required Reading:

pp. 41-55; 725-746 in FOB
pp. 100-110 in MCB

Suggested Questions to Answer:

Study Exercises 1-3 (review), 4&5 and Problem 2 on p. 75 of FOB
Study Exercises 1, 2, 4, 5 and Problems 2, 6, 7 on p. 771 of FOB
Problems 5 & 6 on p. 135 of MCB

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

At least two additional processes are required for gene expression (the functional manifestation of genetic information):

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

Nucleic acids play two kinds of roles in these processes:

Informational

DNA and RNA are uniquely suited as repositories for the storage/retrieval of genetic information
Both molecules serve primarily or exclusively as templates (DNA directs DNA & RNA synthesis, mRNA directs protein synthesis)
DNA and mRNA are not part of the catalytic machinery for these processes
The sequence per se is what really matters: 3-D structures are of secondary importance

Dynamic

rRNAs are integral parts of the ribosome - direct catalysis of peptide bond synthesis
tRNAs are central to translation: provides means for decoding mRNA
Additional RNAs function in splicing/ RNA processing
Certain sequences in DNA and (m)RNA function as recognition sites for components that regulate gene expression

II. Review of Nucleotide Structure (see Figs 4-1, 4-2 in MCB)

Bases

Purines and pyrimidines
Common ring atoms, numbering
Variable substituents, H-bonding potential (donor/acceptor) (Fig. 23-1, FOB)
Tautomeric shifts and effects on H-bonding (Fig. 3-7, FOB)

Nucleosides

Base + sugar (ribose or deoxyribose) linked by N-glycosidic bond (Fig. 23-5, FOB)
1'-carbon of (deoxy)ribose (beta anomer)
Ring N of base (#1 - pyrimidines, # 9 - purines)

Nucleotides:

Nucleoside monophosphates: phosphoester bond links P to sugar (usu. 5' carbon)
Nucleoside di- and triphosphates: additional P linked via high energy phosphoanhydride bond(s)

Nomenclature (Table 3-1, FOB or 4-1, MCB)

bases: adenine, cytosine, guanine, thymine, uracil
nucleosides: (deoxy)adenosine, (deoxy)cytidine, (deoxy)guanosine, thymidine, uridine
nucleotides - (d)NMPs (nucleoside monophosphates)

(d)AMP, (d)CMP, etc
(deoxy)adenosine monophosphate, (deoxy)cytidine monophosphate, etc.
adenylate, cytidylate, guanylate, thymidylate, uridylate

(d)NDPs and (d)NTPs:

GDP=guanosine diphosphate, dATP = deoxyadenosine triphosphate, etc.

DNA and RNA

ribose (RNA) vs. 2'-deoxyribose (DNA)
Thymine/thymidine (DNA) vs. uracil/uridine (RNA)
DNA is typically double-stranded (ds), RNA is usu. single-stranded (ss)
Relative size: DNA usually enormous/continuous, RNA comes in smaller, discrete molecules
Chemical stability: RNA is subject to base hydrolysis (2í OH acts catalytically)

Functions of nucleotides

Energy carriers: drive molecular pumps & motors; link anabolism & catabolism
Cell regulation and signaling (e.g., cAMP; ppGpp in bacteria)
Coenzyme structure (NAD, FAD, Coenzyme A)
Carriers of "activated" molecules (e.g., UDP-sugars for polysaccharide synthesis)
Precursors/building blocks for DNA and RNA

III. Covalent (primary) structure of Nucleic acids (Fig. 3-6, FOB)

Condensation" of (deoxy)nucleotides (NMP residues) into linear chains
Adjacent nucleotides are linked by phosphodiester bonds
Like proteins/polypeptides, nucleic acids have a linear sequence with:

Constant "backbone" (ribose-phosphate)
Variable "side-chains" (bases)
Polarity: 5' end is distinct from 3' end
(Phosphate(s) often present at 5' end; 3' end typically has free -OH group)

By convention, base (nucleotide) sequences are specified from 5' to 3':

Direction of synthesis for both DNA and RNA
Direction in which sequences are read/decoded during protein synthesis (translation)

The structure & function of a DNA or RNA molecule is determined by its sequence of BASES

--> so, why are these "nucleic acids", rather than "nucleic bases"?

Phosphate carries negative charges (~1 per nucleotide at pH7)

Positive charge is negligible: the purine/pyrimidine "bases" are not very basic

Note: In biology/biochemistry, "acidic" and "basic" have somewhat different meanings than in chemistry. To a biochemist, molecules or functional groups are classified based on whether they are - or have the potential to become - negatively-charged (acids) or positively-charged (bases). While chemists usually make a distinction between the "acid" (proton donor) acetic acid and the "base" (proton acceptor) acetate, both are "acidic" under the biochemical definition, which recognizes these as different states of what is the essentially the same molecule. Ultimately, the two definitions match if one considers only the NEUTRAL (non-ionized) form of a molecule or functional group. However, note that for a typical acidic or basic group, the ionized form may prevail over the neutral form under physiological conditions (pH ~ 7). Paradoxically, this means that in the cell, negatively charged "acids" (e.g., DNA) exist primarily as (conjugate) bases, while positively charged "bases" (e.g., protonated amines/amino groups) are actually potential proton donors (acids in the chemical sense).

IV. Higher order structure of DNA: the Double Helix

Antiparallel: 2 strands have opposite 5'-3' orientations (Fig. 4-4b, MCB)
Helix is right handed (except Z DNA) (Fig. 4-4a, MCB)
Base Complementarity: bases are ìinsideî helix and must match: G to C, A to T (Fig. 23-1, FOB)

Chargaffís rules: for dsDNA, G/C and A/T ratios = 1, purines = pyrimidines
G+C vs. A+T content is variable
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 ñ other strand is implied

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

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

Most DNA approximates the ìBî form proposed by Watson and Crick

10 bp/turn (so 36 degrees per bp) ñ 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 2í-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

V. Secondary structure: base pairing in ssRNA (or ssDNA) (Fig. 4-12, 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 ìpalindromicî 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.:

5í ..GCGAACGÖÖÖCGTTCGC.. 3í

3í ..CGCTTGCÖÖÖGCAAGCG.. 5í

Palindromes may be ~perfect (hairpins) or interrupted (stem-loops)
Helical regions may be imperfect due to:

Unusual (non-Watson-Crick) base pairs
Unmatched bases on one strand

VI. 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 (but not pairing) also occurs in ssDNA/RNA

Ionic interactions:

Intrinsic repulsion between negatively-charged phosphates
Stabilized by counterions in solution: mono- and 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. 4-9, MCB)

Relatively sharp transition indicates cooperativity (ìall or noneî)
T_m = temp. required for 50% denaturation (at equilibrium)

Stability (melting temperature):

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

Renaturation:

Requires temp. below (but near) T_m
Basis for various hybridization methods (Southern blotting, cloning, etc)