Chapter 9: Molecular Structure of DNA and RNA

Our knowledge of genetics comes from…

Our knowledge of the molecular structure of DNA and RNA

Who discovered nuclein?

chemist Friedrich Miescher

In a 1971 essay in the history of nucleic acid research, Erwin Chargaff noted that in a 1961 historical account of nineteenth-century science, who was and was not mentioned?

Mentioned: Charles Darwin (31 times) and Thomas Huxley (14 times)

Not Mentioned: Friedrich Miescher (not even once)

How long would DNA actually stretch out?

DNA would actually stretch to the sun and back 4 times!

The strands of DNA are so fine, what is needed to see the DNA strands?

A high power electron microscope

The human genome, the genetic code in each human cell contains how many sets of chromosomes?

23 sets of chromosomes

How Manu nucleotide pairs can each DNA molecule contain? And what’s is their size when uncoiled?

500 thousand to 2.5 million nucleotide pairs

-DNA molecules of this size are 1.7 to 8.5cm long when uncoiled, or about 5cm on average.

How many miles would DNA be able to stretch out end to end?

DNA would stretch out over 744 million miles.

-Variation is due to the estimated number of cells in the body.

What is the percentage of the human genome that contains DNA that helps “build” us or that “maps for proteins”?

1.5%

What is the percentage of DNA that is considered “junk: or control components that we are not sure how it is used?

98.5%

What are the four roles that genetic material must meet to fulfill its role?

1. Information: It must contain the information necessary to make an entire organism.

2. Transmission: It must be passed from parent to offspring.

3. Replication: It must be copied.

   • In order to be passed from parent to offspring.

4. Variation: It must be capable of changes.

   • To account for the known phenotypic variation in each species.

Griffith’s Transformation Experiment

Used two strains of Streptococcus Pneumoniae (pneumococcus)

Griffiths’s Transformation Experiment

• S strain

Infectious (virulent) and has a polysaccharide coat. Infection will result in the death of the animal. Two substrings: IIS and IIIS

Griffiths’s Transformation Experiment

• R strain

Noninfectious (avirulent) and has no polysaccharide coat.

One out of 10^7 S cells will mutate into R.

Ex: type IIS into IIR. However, the reverse is very rare (only IIR into IIS never into IIIS)

The agent involved in Griffiths’s Transformation Experiment was called what and why?

The Transforming Principle because Griffith believed that the transforming principle was the protein.

• We now know that the Transforming Principle is DNA

How was it determined that the transforming principle was DNA instead of protein?

A chemical method (A modern DNA mini prep) was used to isolate chemically pure transforming principle from lazed type IIIS bacteria cells.

This procedure separates the lysate into various cellular macromolecular components: lipids, polysaccharides, proteins, DNA and RNA.

1944: The experiments of Avery, MacLeod and McCarty

Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material.

• At that time, it was known that DNA, RNA, proteins and carbohydrates are the major constituents of living cells.

What did Avery and colleagues isolate during 1944?

They isolated DNA and RNA and tested with crude extracts of nucleases.

• This demonstrates that the transforming principle is DNA.

• However, data was criticized because the DNA was not pure. It was contaminated with protein.

What was the answer Hershey and Chase found in 1952?

Using bacteriophage to confirm that the transforming principle was DNA. DNA is in the head of the viral particle, which is made of protein.

What is the life cycle of a virulent phage, such as T2?

1. Attachment of phage to E.coli and injection of phage chromosome.

2. breakdown of bacterial chromosome by phage-specific enzyme.

3. Replication of phage chromosome using bacterial materials and phage enzymes.

4. Expression of phage genes to produce phage structural components.

5. Assembly of progeny phage particles.

6. Release of progeny phages by lysis of bacterial wall.

Hershey-Chase experiment demonstrating DNA is genetic material:

DNA and RNA are considered…

Large macromolecules with several levels of complexity.

What do nucleotides form?

Nucleotides form the repeating unit of nucleic acids. Nucleotides are linked to form a linear strand of RNA or DNA.

• Two strands can interact to form a double helix.

How are 3D structures of DNA created?

The 3D structure of DNA results from folding and bending of the double helix. Interaction of DNA with proteins produces chromosomes within living cells.

What is a nucleotide?

The nucleotide is the repeating structural unit of DNA and RNAW

What are the three components of a nucleotide?

1. A phosphate group

2. A pentose sugar

   • Ribose in RNA

   • Deoxyribose in DNA

3. A nitrogenous base

Why are they called nucleic acids?

1. They are found in the nucleus

   1. They are acidic

Deoxyribonucleotide

• dAMP

• dCMP

• dGMP

• dTMP

Ribonucleotide

• AMP

• GMP

• CMP

• UMP

Base + Sugar is a

nucleoside

What are examples of a nucleoside?

• Adenine + ribose = Adenosine

• Adenine + deoxyribose = Deoxyadenosine

Base + sugar + phosphate(s) is a

Nucleotide

What are examples of a nucleotide?

• Adenosine monophosphate (AMP)

• Adenosine diphosphate (ADP)

• Adenosine triphosphate (ATP)

Nucleotides are covalently linked together by?

Phosphodiester bonds

• A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of another.

Does a DNA strand have directionality?

Yes the strands have directionality

• 5’ to 3’

• In a strand, all sugar molecules are oriented in the same direction

What forms the backbone of the nucleic acid strand?

The phosphates and sugar molecules, the bases project from the backbone.

What did James Watson and Francis Crick do?

They elucidated the double helical structure of DNA.

• The scientific framework for their breakthrough was provided by other scientists including:

  • Linus Pauling

  • Rosalind Franklin and Maurice Wilkins

  • Erwin Chargaff

Linus Pauling

Proposed that regions of protein can fold into a secondary structure, the alpha helix

• To elucidate this structure, he built ball-and-stick models

Rosalind Franklin

Performed X-ray diffraction of DNA fibers.

• She worked in the same lab as Maurice Wilkins

• She used X-ray diffraction to study wet fibers of DNA

  • The diffraction pattern is interpreted (using mathematical theory)

  • This can ultimately provide information concerning the structure of the molecule

What did Rosalind Franklin make?

She made marked advances in X-ray diffraction techniques with DNA.

What are the suggested structural features of DNA that Rosalind Franklin obtained from the diffraction pattern?

• Helical

• More than one strand

• 10 base pairs per complete turn

Erwin Chargaff

He pioneered many of the biochemical techniques for the isolation, purification, and measurement of nucleic acids from living cells.

He analyzed the base composition of DNA isolated from many different species.

It is known that DNA contains the four bases:

A,G,C,T

The Goal (Discovery-based science)

An analysis of the base composition of DNA in different species may reveal important features about the structure of DNA.

Chargaff’s rules state

that DNA from any cell of any organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount of guanine should be equal to cytosine and the amount of adenine should be equal to thymin.

James Watson and Francis Crick

Published the Double Helix model for the DNA structure.

The Double Helix model was based on the following data:

1. DNA was composed of bases, sugars, and phosphates which were linked as a polynucleotide.

2. The amount of A=T and G=C

3. The ratio of (A+T)/(G+C) varies with organisms.

4. X-ray diffraction analysis indicated:

   1. DNA was in a highly ordered helical structure with distinctive regularities of 0.34nm

The two strands of DNA run…

Anti-parallel, one runs in the 5’ to 3’ direction and the other 3’ to 5’

Two strands are twisted together around a common axis. How many bases complete a turn of the helix?

There are 10 bases and 3.4nm per complete turn of the helix.

The helix is

right handed, as it spirals away from you the helix turns in a clockwise direction.

The double bonded structure is stabilized by:

1. Hydrogen bonding between complementary bases.

   1. A bonded to T by two hydrogen bonds

   2. C bonded to G by three hydrogen bonds

2. Base stacking

   1. Within the DNA, the bases are oriented so that the flattened regions are facing each other

There are two asymmetrical grooves on the outside of the helix

1. Major groove

2. Minor groove

Certain proteins can bind within these grooves

They can thus interact with a particular sequence of bases

The DNA double helix can form different types of

secondary structure

The predominant form found in living cells is

B DNA, however under certain conditions, Z DNA double helices can form

B DNA

Bases relatively perpendicular to the central axis

Z DNA

Bases substantially tilted relative to the central axis

Sugar-phosphate backbone follows a zigzag pattern

Z DNA function in Cell

• left-handed helix

• 12 bp per turn

• Its formation is favored by

  • Alternating purine/pyrimidine sequences, at high salt concentrations

  • Cytosine methylation, at low salt concentrations

  • Negative supercoiling

• May play a role in transcription and chromosome structure

  • Recognized by cellular proteins

  • May alter chromosome compaction

Alexander Rich

discovered triplex DNA

• It was formed in vitro using DNA pieces that were made synthetically

how is a triplex DNA formed?

It was discovered that natural double-stranded DNA can join with a synthetic strand of DNA to form a triplex DNA

• The synthetic strand binds to the major groove of the naturally- occurring double-stranded DNA

Triplex DNA formation is sequence specific

T binds to an AT pair in biological DNA

C binds to a CG pair in biological DNA

Triplex DNA has been implicated in several cellular processes

Recombination, inactivation of specific genes

Potential tool for therapeutic gene inactivation

In RNA synthesis

only one of the two strands of DNA is used as a template

Although usually single-stranded, RNA molecules can form short double-stranded regions

This secondary structure is due to complementary base-pairing

A to U and C to G

Allows short regions to form a double helix

RNA double helices typically

Are right handed

have the A form with 11 to 12 base pairs per turn

Different types of RNA secondary structures are possible

a. Bulge loop

b. Internal loop

c. Multibranched loop

d. Stem loop or Hairpin loop

Many factors contribute to the tertiary structure of RNA

• Base-pairing and base stacking within the RNA itself

  • Interactions with ions, small molecules and large proteins

Xist

long noncoding RNA (IncRNA)

The Xist RNA, a large (17kb in humans) transcript, is expressed on the inactive chromosome and not on the active one.

The transcript remains in the nucleus where it coats the inactive X chromosome