The structure and character of the nucleic acid

School of Life Science

Shandong University

Ch2 &6: The structure and character of the nucleic acid

1. Nucleic Acids convey genetic information

2. DNA structure

3. DNA topology

4. RNA structure

What were the hereditary factors that transmitted the different traits from one generation to the next?

1. Nucleic Acids convey genetic information

The molecules should be stability that the gene demanded, yet be sufficient complexity to convey genetic information, be capable of permanent, sudden change to the mutant forms that must provide the basis of evolution.

1.1 Experiment of Transformation of Streptococcus pneumoniae(肺炎链球菌)

——from 1928-1944，a story written for 16 years

There are 2 forms of S. pneumoniae.

smooth: the bacteria have a polysaccharide capsule（多糖的荚膜） that protects them from the immune system. It also gives the colonies a smooth appearance.

rough: the bacteria lack the capsule and are destroyed by the immune system. They produce rough colonies.

1.1. DNA can carry genetic specificity

Streptococcus pneumoniae

Figure 2-1 Transformation of a genetic characteristics of a bacterial cell by addition of heat-killed cells of a genetically different strain.

严谨的实验设计

——DNA是主要的遗传物质

Figure 2-2 Isolation of a chemically pure transforming agent

1.2. Viral genes are also nucleic acids

2.1. DNA is composed of two polynucleotide chains （多聚核苷酸链）

DNA is composed two polynucleotide chains are twisting around each other in the form of a double helix

p102 Figure 6-1

2. DNA structure

The monomers in polynucleotide chains are nucleotides.

Each nucleotide has 3 parts:

a 5 carbon sugar

a nitrogen base

a phosphate

Formation of nucleotide by removal of water

dehydration synthesis 脱水合成

p103 Figure 6-2

糖苷键 磷酸酯键

The composition and sequence of the base pairs affects what type of structure the DNA forms

sugar phosphate backbone

phosphodiester bond

When you write the sequence of a piece of DNA, you always begin with the 5’ end.

(c)

(d) CAG

2. 2 Each base has its preferred tautomeric form

Each of the bases can exist in two alternative tautomeric states. These states are in equilibrium.

Most of the time the base is in the normal state. Only rarely are the altered forms present.

The altered forms can form different hydrogen bonds, so tautomerization can cause mutations during DNA synthesis

Amino imino (亚氨基)

keto enol

p105 Figure 6-5

p104 Figure 6-3

2.3. The two strands of the Double Helix are held together by base pairing in an antiparallel orientation

This antiparallel orientation a stereochemical (立体化学的) consequence of the way that A-T and G-C pair with each other.

The size of an A:T pair is the same as the

size of a G:C pair.

This means that both can fit in the space

between the sugar-phosphate backbones.

2.4. The two chains of the double helix have complementary sequences

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.

“Waston-Crick” pairing

P106 Figure 6-6

Thymine Adenine

Guanine Cytosine

2.5. Hydrogen Bonding Is Important for Base Pairing

Adenine hydrogen bonds to thymine. It does not form hydrogen bonds with cytosine

Thus, the adenine always binds to thymine and never to cytosine. The hydrogen bonds determine which bases will pair with each other

p107 Figure 6-7

2.6. Base can flip out from double helix

It is energetically favorable for bases to form hydrogen bonds in DNA

However, sometimes a single base pair will move to stick out the side of the double helix. This is called base flipping (碱基外掷)

Base flipping is important in some systems of DNA repair

p107 Figure 6-8

2.7. DNA is usually a right-handed double helix

Please read the Box6-1， p108

p107 Figure 6-9

2.8. The double helix has minor and major grooves

Why？

--geometry几何学

180

270

2.9. The major groove is rich in chemical information

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.

These patterns of hydrogen bonding and shapes allow proteins to bind to specific DNA sequences. Amino acids in a protein can form noncovalent bonds with the base pairs in the major groove.

A: H-bond acceptors D: H-bond donors H: non-polar hydrogens M: methyl groups

p109 Figure 6-10

2.10. The double helix exists in multiple conformations

DNA can exist in 3 different structures:

A DNA B DNA Z DNA

p111 Figure 6-11

Under physiological conditions, DNA is generally in the B form. It has 10 base pairs per turn and major and minor grooves.

When there is less water available and binds to some proteins, DNA is often in the A form. it has 11 base pairs per turn, so it is more compact.

Z DNA forms when the DNA sequence has alternating purines and pyrimidines and there are high concentrations of positive ions (such as Na+) that form ionic bonds with the negative phosphates. It has 12 base pairs per turn. And it has a left-handed helix .

The between the purine and pyrimidine base pairs of a right-hand helix.

p111 Figure 6-12

propeller twist normal

There are two ways the base connects to the deoxyribose. The N-glycosylic bond can be in the syn or the anti conformation.

反式

顺式

p113 Figure 6-13

2.11. DNA sometimes form a left-handed helix

2.12.DNA strands can separate and reassociate（denaturation and Renaturation）

Native: the normal structure of a nucleic acid in nature. Denaturation (Melting，熔解): A process of the hydrogen bonds in DNA were broken which cause the two strands separate. ----heat, high pH, etc Renaturation (Annealing，退火) : A process of the single strands meet their complementary strands and re-form regular double helices. Hybridization: The capacity of single DNA strands to re-form artificial hybrid DNA molecules from two different sources ----complementary sequence techniques！

The absorbance at 260nm for diferent nucleotides

When the Double stranded DNA has an absorbance (A260) of 1.0, the same concentrate:

Single stranded DNA has an absorbance (A260) of 1.37.

Free nucleotides have an absorbance (A260) of 1.60.

RNA have an absorbance (A260) higher than DNA

Purity：A260/A280:

dsDNA--1.8； pure RNA--2.0；protein--0.5

共轭双键

When the temperature of a solution of DNA is raised above its melting temperature (usually more than 80 oC), the double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light. The absorbance at 260 nm markedly increases, the phenomenon is known as hyperchromic.

hyperchromicity—增色效应

This reflects the unwinding of the DNA double helix, since the stacked base pairs absorb less light

When the temperature is lowered, the absorbance decreases, reflecting that the DNA is again stacking.

One way to study the denaturation of DNA is to measure its absorbance of UV light.

变性过程中DNA构型的变化规律

RNA: the absorbance increases gradually and irregularly

DNA: the absorbance increases cooperatively.

Plotting A260 versus temperature on a graph gives you a melting curve.－溶解曲线

The melting temperature (Tm) is defined as the point where half of the DNA is denatured.

Tm is 85oC

The melting temperature is a characteristic of each DNA——Tm值是DNA的特征常数，与下面的因素有关。

1) Tm depends on G·C percentage. the higher the G·C percentage, the greater the Tm. (more energy is required to break the extra hydrogen bonds).

2) Tm depends on DNA length. the longer the DNA, the greater the Tm

3) Effect of [Salt] on Tm，the higher the salt concentration of the solution, the greater the Tm

For oligos 4-18 nucleotides Tm = (A+T) 2 ℃ + (G+C) 4 ℃

Increasing G-C content

Pneumococcus 38% G+C

E.coli 52%

S. marcescens 58%

M.phlei 66%

Temperrature (℃)

40% 51%

73%

low salt

high salt

p107 Figure 6-7

dsDNA和ssDNA表现出不同的变性曲线

Why？

When DNA is heated it denatures and the two strands separate.

Then when the DNA is slowly cooled the complementary strands renature.

Renaturation

复性条件：

For renaturation to occur the DNA must be at the right temperature ①— one that allows some base pairing to occur but that is high enough to prevent single stranded DNA from binding to itself. (破坏链内氢键的形成)

The optimal temperature is based on the G-C content② of the DNA and is about 5o C below the melting temperature (Tm) ——8-18nt

Renaturation also requires the correct salt concentration③. The negatively charged phosphates in the DNA backbone repel each other. Na+ or Mg++ ions bind to the phosphates and neutralize the charges.

0.15-.5M NaCl works well

Renaturation occurs best at high concentrations of DNA ④ .

Renaturation requires time⑤for the complementary strand to pair. Longer time allows more annealing to occur up to a point.

复性机制：

At the molecular level renaturation occurs in 2 steps. First there is an initial slow step of base pair binding often called nucleation(成核作用). If the DNA is complementary, the second step which is often called zippering is fast.

复性过程是一个随机混合的过程

In renaturation a DNA strand will hydrogen bond to a complementary DNA strand in solution. This will likely not be the original DNA strand that it was bound to before it was denatured. 大部分复性分子都不是原配的。

Similar to measuring

denaturation, renaturation can also be studied by measuring A260. As the strands of DNA renature, the absorbance will decrease.－－减色效应

复性分数 C/C0 是C0t的函数，

这函数曲线称为C0t曲线。 ——C指单链DNA的浓度

复性分数

起始浓度X时间,C0t

C/C0

① DNA复性过程

遵循二级反应动力学

复性速度常数

DNA序列的复杂性影响k值

5 kb Repeats (20% total)

100 kb fragment

Repeat DNA = 5 kb

Longest non-repeat DNA = 100kb - 20 kb = 80 kb

Difference in complexity = 80/5 = 16

DNA with many repeated sequences will renature much faster than DNA with no repeated sequences.

DNA序列的复杂性（complexity）X: 最长的没有重复序列的核酸对的数值

Eukaryotic genomes have several sequence components

重复度

② 复性与DNA的（复杂程度）

重复序列有关，组分复性越快复杂性越低。

Explanation of complexity

C0t values are directly proportional to the

complexity of the genome.

1－C/C0

2.13.Some DNA molecules are circles

Examples:

• The chromosome of the small monkey DNA virus SV40.

• Most bacterial chromosomes.

• Plasmids: The small autonomously replicating genetic elements outside of chromosomes.

3. DNA topology

At the time of the discovery of the double helix structure of DNA, scientists thought all DNA molecules were linear and had two free ends.

But there were some circular DNA molecules in nature (plasmid, bacteria chromosome, etc).

The ends of linear DNA can freely rotate to adjust to changes in the number of times the strands are twisted around each other, but in circular DNA this is not possible.

In circular DNA the number of times the DNA strands are twisted around each other cannot change.

Closed circular DNA cannot unwind. It is topologically constrained.

Species of cccDNA： Plasmid and circular bacterial chromosomes Linear DNA molecules of eukaryotic

chromosomes due to their extreme length, entrainment (缠卷) in chromatin and interaction with other cellular components (Ch 7)

3.1. Linking number is an invariant topological property of covalently closed, circular DNA (cccDNA，共价闭合环状DNA)

Linking number (交叉数、环绕数) ------ 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.

一条链绕另一条链环绕的次数（Lk）

Lk0 is the linking number of fully relaxed cccDNA under physiological conditions

Linking number=0

Linking number=1

Linking number=2

The linking number is the sum of the twist (扭曲)and the writhe（翻腾）.

Twist(Tw)is the number of times one strand completely wraps around the other strand.

Writhe(Wr) is the number of times that the long axis of the double helical DNA crosses over itself in 3-D space.

3.2. Linking number(Lk) is composed of Twist and Writhe

Lk= Tw + Wr

There are 2 kinds of writhe:

Interwound Spiral盘旋

Please read p118－120 for detail

互相盘绕的

Relaxed Closed circular DNA

Local disruption of base pairing

Negatively Superoiled

p118 Figure 6-17

3.2. Lk0 is the Linking number of fully relaxed cccDNA under physiological conditions

• Lk0 for such a molecule is the number of base pairs divided by 10.5. Since in a relaxed state in solution the two polynucleotide strands wrap around each other such that there are 10.5 base pairs per turn. For a cccDNA of 10,500 base pairs, Lk=+1000.

• If there are more or less than 10.5 base pairs per turn, the DNA is supercoiled.

Adding twists to the DNA causes it to be supercoiled or superhelical. 增旋而形成的超螺旋为正超螺旋

Unwinding the DNA causes a negative superhelix. 解旋而形成的超螺旋为负超螺旋

3.3 DNA in cells is negatively supercoiled

• Circular DNA molecules purified from both bacteria and eukaryotes are usually negatively supercoiled, having values of of about -0.06.

ơ =(Lk-Lk0)/Lk0

• The only organisms that have been found to have positively supercoiled DNA are certain thermophiles, microorganisms that live under conditions of extreme high temperatures.

3.4. Topoisomerases can relax supercoiled

Topoisomerases are enzymes that can change the linking number of DNA by making single or double stranded nicks.

There are 2 kinds of topoisomerases:

Type I make a single stranded cut in the DNA.

Type II make a double stranded cut in the DNA.

Type I (Topo I)-⑴每次只切开一条链，⑵每次改变一个超螺旋，⑶反应不需要能量，

Type II (Topo II)- ⑴每次切开两条链，⑵每次改变2个超螺旋，⑶需要能量， Topo II can catenate and decatenate cccDNA molecules.

Properties of the topoisomerase：

P124Figure 6-24

3.5. Topoisomerases cleave DNA using a covalent tyrosine-DNA intermediate

Properties of the topoisomerase：

3.6. DNA topoisomers can be separated by electrophoresis

Relaxed or nick DNA

The speed of the DNA molecules migrate increases as the number of superhelical turns increases

p125 Figure 6-26

4. RNA Structure

4.1. RNA contains ribose and uracil and is usually single-stranded

Sequence complementarity: inter- and intra-molecular base pairing

4.2. RNA chains fold back on themselves to form local regions of double helix similar to A-form DNA

hairpin

bulge

loop

Because RNA is single stranded, it often will base pair to itself between short segments of complementary sequences, which adopt one of the various stem-loop structures

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

Unconventional base pairing, such as base triples, base-backbone interactions in RNA.

A example of hydrogenbonding that allows unusual triples base pairing

tetraloop(UUCG)---- Special interactions

1

2

3

4

水平线： 碱基堆积作用

U:G对

Pseudoknot

4.3. RNA can fold up into complex tertiary structures

The base pair in the single stranded produce the secondary (base pairing) and tertiary (3 dimensional conformation) of the RNA molecule. Pseudoknots （“假结”）are complex secondary structure resulted from base pairing of discontiguous RNA segments.

4.4. Some RNAs are enzymes ---- Ribozymes

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

Please read p130－132 for detail

The hammerhead ribozyme cleaves RNA by formation of a 2’,3’ cyclic phosphate

tertiary structures of the hammerhead ribozyme

a) RNA is the genetic material of some viruses b) RNA functions as the intermediate (mRNA) between the

gene and the protein-synthesizing machinery. c) RNA functions as an adaptor (tRNA) between the codons

in the mRNA and amino acids. d) Through sequence complementarity, RNA serves as a

regulatory molecule to bind to and interfere with the translation of certain mRNAs; or as a recognition molecule to guide many post-transcriptional processing steps.

e) Through the tertiary structures, some RNAs function as enzymes to catalyze essential reactions in the cell.

Biological roles of RNA

Did life evolve from an RNA world?

Thinking-----