CHAPTER 6, Mol. Biol. Gene; Watson et. al.

The Structures of DNA and RNA

How do the structures of DNA and RNA account for their functions?

OUTLINE
1.DNA Structure 2.DNA Topology

3.RNA Structure

DNA STRUCTURE

DNA is composed of polynucleotide chains

Structure: twisting around each other in the form of a double helix.

DNA STRUCTURE ( 1 )

Schematic model

Space-filling model

Nucleoside & Nucleotide, the fundamental building block of DNA

phosphoester bond glycosidic bond

Phosphodiester linkages: repeating, sugar-phosphate backbone of the polynucleotide chain

DNA polarity: is defined by the asymmetry of the nucleotides and the way they are joined.

Bases in DNA

adenine

purines
guanine

N9

cytosine

pyrimidines
thymine
N1

Each bases has its preferred tautomeric form

DNA STRUCTURE ( 2 )

Handedness in DNA

A POLYMER OF DEOXYRIBONUCLEOTIDES DOUBLE-STRANDED INDIVIDUAL deoxyNUCLEOSIDE TRIPHOSPHATES ARE COUPLED BY PHOSPHODIESTER BONDS

A DOUBLE HELICAL STRUCTURE

ESTERIFICATION LINK 3 CARBON OF ONE RIBOSE WITH 5 C OF ANOTHER TERMINAL ENDS : 5 AND 3

COMMON AXIS FOR BOTH HELICES HANDEDNESS OF HELICES ANTIPARALLEL RELATIONSHIP BETWEEN 2 DNA STRANDS

Mica experiment

Each base pair is twisted from the pervious one by 36 degrees needing 10 bases to complete a

BASE STACKING

PARTIAL OVERLAP OF PURINE AND PYRIMIDINE BASES IN SOLID-STATE (CRYSTAL)

VANDERWAALS FORCES MOSTLY HYDROPHOBIC FORCES ENTHALPICALLY-DRIVEN ENTROPICALLY-OPPOSED OPPOSITE TO THAT OF PROTEINS

IN AQUEOUS SOLUTION

HYDROGEN BONDING

REQUIRED FOR SPECIFICITY OF BASE PAIRING NOT VERY IMPORTANT IN DNA STABILIZATION HYDROPHOBIC FORCES ARE THE MOST IMPT.

The two strands of the double helix are held together by base pairing in an antiparallel orientation, which is a
stereochemical consequence of the way that adenine and thymine,and guanine and cytosine, pair with each other. (Related to replication

DNA STRUCTURE ( 3 )

and transcription)

The Two Chains of the Double Helix Have Complementary Sequences Watson-Crick Base Pairing Example: If sequence 5-ATGTC-3 on one chain, the opposite chain must have the complementary sequence 3TACAG-5 (Related to replication and transcription)

DNA STRUCTURE ( 4 )

The strictness of the rules for Waston-Crick pairing derives from the complementarity both of shape and of hydrogen bonding properties between adenine and thymine and between guanine and cytosine.

A:C incompatibility

Hydrogen Bonding Is Important for the Specificity of Base Pairing

The hydrogen bonds between complementary bases contribute to the thermodynamic stability of the helix and the specificity of base pairing Stacking interactions between bases significantly contribute to the stability of DNA double helix

DNA STRUCTURE ( 5 )

GEOMETRY OF B-DNA

IDEAL B-DNA HAS 10 BASE PAIRS PER TURN BASE THICKNESS

AROMATIC RINGS WITH 3.4 A THICKNESS TO RINGS

PITCH = 10 X 3.4 = 34 A PER COMPLETE TURN AXIS PASSES THROUGH MIDDLE OF EACH BP MINOR GROOVE IS NARROW MAJOR GROOVE IS WIDE IN CLASS EXERCISE: EXPLORE THE STRUCTURE OF B-DNA. PAY SPECIAL ATTENTION TO THE MAJOR, MINOR GROOVES

Concept of grooves in DNA

The double helix has Minor and Major grooves (What & Why)
It is a simple consequence of the geometry of the base pair.

DNA STRUCTURE ( 5 )

The Major groove is rich in chemical information (What are the biological relevance?)
The edges of each base pair are exposed in the major and minor grooves, creating a pattern of hydrogen bond donors and acceptors and of van der Waals surfaces that identifies the base pair.

DNA STRUCTURE ( 6 )

A: H-bond acceptors

D: H-bond donors

H: non-polar hydrogens

M: methyl groups

The double helix exists in multiple conformations.

The B form (10 bp/turn), which is observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions A form (11 bp/turn), which observed under the condition of low humidity, presents in certain DNA/protein complexes. RNA double helix adopts a similar conformation.

DNA STRUCTURE ( 7 )

DNA STRUCTURE ( 8 )

DNA strands can separate (denature) and reassociate (anneal)

Key terms to understand 1. Denaturation 2. Hybridization 3. Annealing/renature 4. Absorbance 5. Hyperchromicity 6. Tm (melting point)

DNA

STRUCTURE (

) 9

DNA

STRUCTURE (

10

DNA

STRUCTURE (

11

DNA

STRUCTURE (

12

DNA TOPOLOGY

Structure (1): Linking number is an invariant topological property of covalently closed, circular DNA (cccDNA)

DNA TOPOLOGY ( 1 )

Linking number is the number of times one strand have to be passed through the other strand in order for the two strands to be entirely separated from each other.

Species of cccDNA 1. Plasmid and circular bacterial chromosomes 2. Linear DNA molecules of eukaryotic chromosomes due to their extreme length, entrainment in chromatin and interaction with other cellular components: topologically constrained

Structure (2): Linking number is composed of Twist and Writhe The linking number is the sum of the twist and the writhe. Twist is the number of times one strand completely wraps around the other strand. Writhe is the number of times that the long axis of the double helical DNA crosses over itself in 3-D space.

DNA TOPOLOGY ( 2 )

Local disruption of base pairs

Function (1): DNA in cells is negatively supercoiled; nucleosomes introduces negative supercoiling in eukaryotes

DNA TOPOLOGY ( 3 )

Negative supercoils serve as a store of free energy that aids in processes that require strand separation, such as DNA replication and transcription. Strand

separation can be accomplished more easily in negatively supercoiled DNA than in relaxed DNA

Function (2): Topoisomerases

DNA TOPOLOGY ( 4 )

1. The biological importance of topoisomerase? 2. The functional difference of the two types of topoisomerases? 3. The working mechanism of topoisomerase

Ethidium Ions Cause DNA to Unwind

Ethidium: large, flat, multi-ringed cation

Intercalates between stacked base pairs

RNA STRUCTURE

RNA contains ribose and uracil and is usually single-stranded

RNA STRUCTURE ( 1 )

Biological roles of RNA

1. RNA is the genetic material of some viruses 2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery. 3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids. 4. RNA serves as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of certain mRNAs. 5. Some RNAs are enzymes that catalyze essential reactions in the cell (RNase P ribozyme, large rRNA, self-splicing introns, etc).

Structure (1): RNA chains fold back on themselves to form local regions of double helix similar to A-form DNA

RNA STRUCTURE ( 2 )

RNA helix are the basepaired segments between short stretches of complementary sequences, which adopt one of the various stem-loop structures

hairpin
bulge

loop

Some tetraloop sequence can enhance the stability of the RNA helical structures
For example, UUCG loop is unexpectedly stable due to the special base-stacking in the loop

Pseudoknots are complex structure resulted from base pairing of discontiguous RNA segments

Figure 6-32 Pseudoknot.

Non-Watson-Crick G:U base pairs represent additional regular base pairing in RNA, which enriched the capacity for self-complementarity

Figure 6-33 G:U base pair

The double helical structure of RNA resembles the A-form structure of DNA.
The minor groove is wide and shallow, but offers little sequence-specific information. The major groove is so narrow and deep that it is not very accessible to amino acid side chains from interacting proteins. Thus RNA structure is less well suited for sequence-specific interactions with proteins

Structure (2): RNA can fold up into complex tertiary structures Why?

RNA STRUCTURE ( 3 )

RNA has enormous rotational freedom in the backbone of its non-base-paired regions

Interactions in the tertiary structure

Unconventional base pairing, such as base triples, base-backbone interactions Proteins can assist the formation of tertiary structures by large RNA molecule

RNA STRUCTURE ( 4 )

Function: Some RNAs are enzymes Ribozymes are RNA molecules that adopt complex tertiary structure and serve as biological catalysts. RNase P and self-splicing introns are ribozymes

Structure & Function: The hammerhead ribozyme cleaves RNA by formation of a 2,3 cyclic phosphate

RNA STRUCTURE ( 5 )

RNA STRUCTURE ( 6 )

RNAse P : composed of both RNA & Protein: generates tRNA from precursors RNA moiety : catalyst Protein moiety: shields ve charges so that it can bind effectively on its substrates Hammerhead: generally found in plants as viroids depends on self cleavage to propagate (replicates)

In Brief ..

Deoxyadenynyl-3',5'-guanylyl-3'-phosphate

Forms of DNA
B- DNA: This is the structure of fully hydrated DNA, and is the most common encountered in vivo. There is about a 6o tilt of the bases to the helix axis and the axis goes through the center of the base pairs. The diameter of B-DNA is 20 A, and there are about 10 bases per helical turn. The major groove is wide, while the minor groove is narrow. A-DNA: When B-DNA is dehydrated, there is a reversible structural change to ADNA, in which there is an increase in the tilt of the bases to about 20o with respect to the helical axis. The result is a structure with a narrow and deep major groove and a wide and shallow minor groove, a diameter of about 26 A, and 11.6 bases per helical turn. When looking at A-DNA end-on, there is a 6-A diameter hole at the helical axis. Z-DNA: Unlike B-DNA and A-DNA, Z-DNA is a left-handed helix. It has a base tilt of about 7o and a diameter of about 18 A. There are 12 bases per helical turn, a narrow and deep minor groove and a flat major groove. Sequences of DNA consisting of alternating purine and pyrimidine bases have been shown to adopt this conformation. Such sequences include poly d(GC), poly d(AC), poly d(GT) etc. in varying combinations and can be formed at high salt concentrations, under dehydrating conditions in the presence of Mn2+ or Co2+ , or in "supercoiled" DNA. The conformational change from B-DNA to Z-DNA is one mechanism for relief of the torsional strain found in B-DNA in vivo, and may serve as a switch mechanism to regulate gene expression