Reading Assignment: Chapter 2, pgs 12-29, Chapter 12, pgs 315-323

DNA & RNA Structure

BCH 5413 Dr. Yang Fall 2013

The Central Dogma - Updated

Transcriptional control

DNA replication

Translational control
Post-translational processing & modification

Transcription

Translation

DNA
Reverse transcription

RNA
RNA processing, editing, catalysis

Protein

Active Protein

Regulation of RNA processing

RNA-mediated regulation of transcription

Nucleic Acid Bases

DNA Trinucleotide
Phosphodiester bonds

*
RNA-DNA Sugars

-- Chargaffs rules: A=T and C=G -- Propeller twist about 1o -- Each base pair presents unique chemical groups to the major groove
Major

Hydrogen Bonding in DNA

Minor

Watson-Crick Base Pairing

Major

Minor

DNA Structure

Semi-Conservative Replication of DNA (The Meselson & Stahl Expt.)

Alternative Double-Stranded DNA Conformations

A-DNA: -- dehydrated form -- right-handed helix -- 11 bases per helical turn -- base pairs tilted 19o -- major groove narrow and deep

A-DNA

B-DNA

Z-DNA

Z-DNA -- >1% of cellular DNA -- favored by G-C repeats -- left-handed helix -- 12 base pairs per helical turn -- base pairs tilted 9o -- major groove flattened, nearly gone -- Poxvirus E3L virulence factor binds Z-DNA down-regulating apoptosis genes

RNA-DNA hybrids RNA double helices

Non-duplex DNA Structures

Cruciform DNA -- inverted repeat sequence -- favored by excessive negative supercoiling -- AT-rich cruciforms associated with fragile DNA
Atomic force microscopy

Pennisi (2006) Science 312, 1467-8

Triplex and Quadruplex DNA Structures Non-Watson and Crick base pairing

Hoogsteen base pairs

Triplex DNA -- pyrimidine-rich strand -- negative supercoiling Chair DNA -- two G-rich strands -- down-regulation of c-myc transcription
Pennisi. 2006. Science 312, 1467

Intrinsic Bends in DNA

Distortion of the ideal B-DNA conformation resulting from base stacking in the nucleotide sequence Example: A-tract DNA results in 20o bends
unwind helix tilt A-tract base pairs stack the base pairs rewind the double helix

Crothers et al (1990) J. Biol. Chem. 265, 7093

Intrinsic Bend in DNA

Duplex-oligonucleotide Model DNA
5 CATGGCCATG 3 3 GTACCGGTAC 5

-- 23o bend of helical axis -- mis-stacking of one GC -- propeller twist of central GC base pairs
DNA is not a uniform structure; many localized variants
Goodsell et al (1993) Proc. Natl. Acad. Sci. 90, 2930

Supercoiled DNA
Left-handed under-twisted DNA is in a Negative Supercoil. Right-handed over-twisted DNA is in a Positive Supercoil.

Electron Microscopy Relaxed Supercoiled

Most biological DNA is negatively supercoiled. Topoisomerases alleviate supercoils in DNA.

Denaturation/Melting of DNA
Separation of DNA strands Tm = 81.5oC + 16.6(log10[Na+]) + 0.41(%G+C) 0.63(% formamide) (600 / l)

[The hyperchromicity effect]

Tm increases with GC content

The greater G+C content and the higher the salt, the more stable the DNA duplex. Higher Stringency conditions are high heat and low salt High pH (i.e., base) also denatures DNA

DNA Renaturation and Hybridization

Renature

(or DNA)

How Do Sequence-Specific DNA-Binding Proteins Bind in a Sequence-Specific Manner?

Zinc Finger Binding Proteins

Custom Designed DNA-Binding Proteins

Triple zinc finger binding in the major groove of DNA

Zinc Fingers (cont.)

Swapping of the -helix and -hairpin regions alters the DNA binding properties of Sp1

DHANASEKARAN et al. Acc. Chem. Res. 2006, 39, 45-52

Zinc Finger Properties

Crystallographic studies
A single zinc finger protein can recognize 3 bp of DNA

Courtesy of J Barrow & J Bungert

(Pabo, Science 1991)

Molecular Pharmacology 66:1361-1371 (2004)

Modular assembly of custom zinc finger proteins. A, crystal structure of three-ZF protein Zif268 bound to DNA (Elrod-Erickson et al., 1996) shows a relatively simple and regular binding pattern of three primary residues at positions -1, 3, and 6 (white, outlined labels) in each finger (gray ribbons) contacting three bases (high-lighted in black, dark gray, and light gray) on one strand of the DNA. Residue numbering is in relation to the start of the -helix. The residue in position 2 often contacts a target site overlap base on the opposite strand (not shown). Spheres represent zinc ions. B, in the modular assembly strategy, recognition modules (boxes) consisting of blocks of seven residues (-1 to 6) are grafted into a regular zinc finger scaffold (C) using standard PCR methods. Some modules exhibit target site overlap (curved dashed lines), requiring a G or T to be present in the neighboring finger's subsite (k following the recognition site [k = G or T]). D, the full set of modules can be grafted in a combinatorial fashion to create multifinger libraries of DNA-binding proteins.

Custom Designer DNA-Binding Proteins!

Potential for designing a DNA-binding protein to bind to a single site (DNA sequence) in the genome

RNA Structure
-- Single-stranded -- 2 OH -- Uracil (not T) -- 5 to 3 orientation Secondary and Tertiary Structure from long range base pairing -- Watson and Crick -- G-to-U -- others

Stem-Loop

Hairpin

Pseudoknot

Secondary Structure of rRNA

16S rRNA

5S rRNA

Tertiary Structure of tRNA

Base pairing and other interactions primarily between D loop and T-psi-C loop convert clover leaf into the L conformation