DNA & RNA Structure

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

BCH 5413

Dr. Yang

Fall 2013

The Central Dogma - Updated

DNA RNA Protein Active Protein Transcription Translation

Post-translational processing & modification

Reverse transcription

DNA replication

RNA processing, editing, catalysis

Transcriptional control

Translational control

Regulation of RNA processing

RNA-mediated regulation of transcription

Nucleic Acid Bases

*

RNA-DNA Sugars

DNA Trinucleotide Phosphodiester bonds

Hydrogen Bonding in DNA

Watson-Crick Base Pairing

-- Chargaff’s rules: A=T and C=G

-- Propeller twist about 1o

-- Each base pair presents unique chemical

groups to the major groove

Major

Minor

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

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

A-DNA B-DNA Z-DNA

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

Triplex DNA

-- pyrimidine-rich strand

-- negative supercoiling

Chair DNA

-- two G-rich strands

-- down-regulation of

c-myc transcription

Hoogsteen base pairs

Non-Watson and Crick base pairing

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

CATGGCCATG

GTACCGGTAC

-- 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

5’ 3’

3’ 5’

Supercoiled DNA

Left-handed under-twisted DNA

is in a Negative Supercoil.

Right-handed over-twisted DNA

is in a Positive Supercoil.

Most biological DNA is negatively

supercoiled.

Topoisomerases alleviate

supercoils in DNA.

Relaxed Supercoiled

Electron Microscopy

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 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

[The hyperchromicity effect]

Tm increases with GC content

DNA Renaturation and Hybridization

Renature

(or DNA)

How Do Sequence-Specific DNA-Binding Proteins Bind

in a Sequence-Specific Manner?

Triple zinc finger binding in the major groove of DNA

Zinc Finger Binding Proteins Custom Designed DNA-Binding Proteins

DHANASEKARAN et al.

Acc. Chem. Res. 2006, 39, 45-52

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

Zinc Fingers (cont.)

• Crystallographic studies

– A single zinc finger protein can recognize 3 bp of DNA

Zinc Finger Properties

(Pabo, Science 1991) Courtesy of J Barrow & J Bungert

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 α-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.

Molecular Pharmacology

66:1361-1371 (2004)

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