

3.3, 7.1 DNA structure
3.4, 7.2 DNA replication
3.5, 7.3, 7.4 Transcription and translation

• Biologists knew that genes are located on chromosomes
(made of DNA and protein)
– DNA and protein were the candidates for the genetic material
–
–
Until the 1940s, the case for proteins seemed stronger because proteins appeared to be more structurally complex and functionally specific.
Biologists finally established the role of DNA in heredity through studies involving bacteria and the viruses that infect them.

•
•
•
•
1928; British medical officer
Griffith was studying two strains of a bacterium:
–
– a pathogenic (disease-causing) strain that cause pneumonia a harmless strain.
Found that when he mixed a dead version of the pathogenic bacteria and harmless bacteria, some living bacterial cells were converted to the disease-causing form.
– Furthermore, all of the descendants of the transformed bacteria inherited the newly acquired ability to cause disease.
Clearly, some chemical component of the dead bacteria could act as a “transforming factor” that brought about a heritable change.





1952; American biologists
Experiments showed that DNA is the genetic material of a virus
(bacteriophage or phage, for short) called T2, which infects E.coli
T2 consists solely of DNA and protein; DNA-containing head and a hollow tail with six jointed fibers extending from it.
T2 infects bacteria by attaching to the surface with its fibers and injecting its hereditary material.
 Raised the question: Is it DNA being passed on or protein?



To answer protein or DNA question, they devised an experiment to determine what kinds of molecules the phage transferred to E.coli during infection
Used a few relatively simple tools:
 Chemicals containing radioactive isotopes


To label the DNA and protein in T2
Used radioactive sulfur and phosphorous
 Sulfur is in proteins. Phosphorous is in DNA.


A radioactivity detector
A kitchen blender
 And a centrifuge (device that spins test tubes to separate particles of different weights.


The Experiment
1.
2.
3.
4.
5.
6.
First they grew T2 with E.Coli with radioactive sulfur.
Then, they grew a separate batch of phages in a solution containing radioactive phosphorous.
They allowed the two batches of T2 to infect separate samples of nonradioactive bacteria
Shortly after the onset of infection, they agitated the cultures in a blended to shake loose any parts of the phages that remained outside the bacterial cells.
They then spun the mixtures in a centrifuge. The cells were deposited as a pellet at the bottom of the centrifuge tubes, but phages and parts of phages being lighter, remained suspended in the liquid.
The researchers then measured the radioactivity in the pellet and the liquid.


The Results


Found that when the bacteria has been infected with T2 phages containing labeled protein, the radioactivity ended up in the liquid but not bacteria.
 Result suggested that the phage protein did not enter the cells.
But when the bacteria had been infected with phages whose DNA was tagged, then most of the radioactivity was in the pellet, made up of bacteria.
 When these bacteria were returned to liquid growth medium, they soon died and lysed and released new phages that contained radioactive phosphorous in their DNA but no radioactive sulfur.


The Conclusions


They concluded that T2 injects its DNA into the host cell, leaving virtually all its protein outside.
They demonstrated that it is the injected DNA molecules that cause cells to produce additional phage DNA and proteins, making new complete phages.


This indicated that DNA contained the instructions for making proteins
***THESE RESULTS CONVINCED MOST SCIENTISTS THAT
DNA IS THE HEREDITARY MATERIAL!!!***

 Deoxyribonucleic Acid (DNA)
 Monomers made up of nucleotides:

 Nucleotides consist of:
 A five carbon sugar, deoxyribose o Four in it’s ring, one extending above the ring

 o Missing one oxygen when compared to ribose
Phosphate group o Is the source of the “acid” in nucleic acid
Nitrogenous base (Adenine, Guanine, Cytosine, Thymine) o A ring consisting of nitrogen and carbon atoms with various functional groups attached o Double ring= purines (A and G) o Single ring= pyrimidines (T and C)
Double helix consists of:


Sugar-phosphate backbone held by covalent bonds
Nitrogen bases are hydrogen bonded together; A pairs with T and C pairs with G





A few scientists working on the puzzle trying to determine the 3-D structure: Pauling, Wilkins, Franklin, Watson and
Crick
Rosalind Franklin observed an X-ray crystallography image of the basic shape of DNA
Watson saw this image, and with just a glance deduced the basic shape of DNA to be a helix with a uniform diameter of
2 nm, with its nitrogenous bases stacked about one-third of a nanometer apart.
The diameter of the helix suggested it was made up of two polynucleotide strands=> DOUBLE HELIX!





Watson and Crick began trying to construct a double helix that would conform both Franklin’s data and what was currently known about the chemistry of DNA.
Franklin had concluded that the sugar-phosphate backbones must be on the outside of the double-helix, forcing the nitrogenous bases to swivel to the interior of the molecule.
Watson and Crick found that Adenine always paired with
Thymine, and Guanine and Cytosine, to ensure a uniform diameter.
Complementary base pairing was explained both by the physical attributes and chemical bonding of DNA, along with data obtained by Chargaff




Chargaff’s rules: A always pairs with T and G always pairs with C.
Only apply to base pairing, not the sequence of nucleotides
 The sequence of bases can vary in countless ways, and each gene has a unique order of nucleotides, or base sequence
1962, Watson and Crick received the Nobel Prize for their work (Franklin would have received it as well, but she died from cancer in 1958; Nobel Prizes are never awarded to the deceased)

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
~Watson and Crick

 Logic behinds Watson-Crick’s proposal for how DNA is copied
 Can be seen by covering one of the strands in the parental DNA molecule with a piece of paper: you can determine the bases of the covered strand by applying the base-pairing rules: A pair with T, and G pairs with C.
 Watson and Crick predicted that a cell applies the same rules when copying its genes.


1.
2.
3.
4.
Figure 10.4A Template model for DNA replication
First, the two strands of parental DNA separate, and each becomes a template for the assembly of a complementary strand from a supply of free nucleotides.
The nucleotides line up one at a time along the template strand in accordance with the base-pairing rules
Enzymes link the nucleotides to form the new DNA strands.
Completed new molecules, identical to the parental molecule, are known as daughter DNA.

 Semi-conservative model



Watson and Crick’s model predicts that when a double helix replicates, each of the two daughter molecules will have one old strand, which was part of the parental molecule, and one new made strand.
Known as semi-conservative model because half of the parental molecule is maintained (conserved) in each daughter molecule.
Confirmed by experiments performed in the 1950s.






DNA replication begins at specific sites on the double helix referred to as origins of replication.
An enzyme called helicase binds to DNA and separates the strands.


Uses energy from ATP
Single-strand binding protein (SSB) binds to each strand to prevent reannealing
Replication proceeds in both directions , creating replication
“bubbles”
DNA has many origins of replication that can start simultaneously, for time efficiency.
Thousands of bubbles can be present, and eventually all the bubbles merge, yielding two completed daughter DNA molecules.





DNA’s sugar-phosphate backbones run in opposite directions.
Each strands has a 3’ end and a 5’ end.
The primed number is referring to the carbon atoms of the nucleotide sugars.
At one end of each DNA strand, the sugar’s 3’ carbon atom is attached to an –OH group, at the other end, the sugar’s 5’ carbon has a phosphate group.




The opposite orientation of the strands is important in DNA replication.
DNA polymerases link DNA nucleotides to a growing daughter strand, only to the 3’ end of the strand, never to the
5’ end.
Thus, a daughter DNA strand can only grow in the 5’  3’ direction.

 Replication fork forms:



Partial opening of a DNA helix to form two single strands that has a fork appearance.
Primers required by DNA polymerases during replication are synthesized by an enzyme RNA primase.
 Enzyme is an RNA polymerase that synthesizes short stretches of RNA that function as primers for DNA polymerases. Later on, the RNA primer is removed and replaced with DNA.
DNA polymerase removes primers and adds DNA nucleotides.
 We will be looking at two types of DNA Polymerase: DNA polymerase
III and DNA polymerase I

 one of the daughter strands can be synthesized in one continuous fashion from an initial primer, working toward the forking point of the parental DNA.
 leading strand
 Only a single priming event is required, and then the strand can be extended indefinitely by DNA polymerase III.

 The other daughter strand polymerase molecules must work outward from the forking point, is synthesized in short pieces as the fork opens up in a discontinous fashion involving multiple priming events.  lagging strand
 DNA Polymerase III adds nucleotides in 5’  3’ direction.
Primers get removed via and replaced by DNA via DNA polymerase I


Fragments formed are called Okazaki fragments
DNA ligase links (ligates) the pieces together into a single DNA strand.



At the completion DNA replication, you end up with two identical strands of DNA.
Each DNA molecule contains one original parent strand, and one new daughter strand.
 Semi conservative replication

 http://www.wiley.com/college/pratt/0471393878/studen t/animations/dna_replication/index.html

 Key enzymes:





Helicase: unwinds the double helix
Primase: synthesizes RNA primers
SSB: stabilizes single-stranded regions; prevents reannealing
DNA polymerase III- synthesized DNA
DNA polymerase I- erases primer and fills gaps
 DNA ligase- joins the ends of DNA segments; DNA repair






Process is not only fast but also amazingly accurate
Typically, only about one DNA nucleotide per billion is incorrectly paired
DNA polymerase carry out a proofreading step that quickly removes nucleotides that have base-paired incorrectly
DNA polymerases and DNA ligase are also involved in repairing damaged DNA by harmful radiation or toxic chemicals
Ensure that all somatic cells in a multicellular organism carry the same genetic information.





DNA inherited by an organism specifies traits by dictating the synthesis of proteins.
However, a gene does not build a protein directly; it dispatches instruction in the form of RNA, which in turn programs protein synthesis.
Message from DNA in the nucleus of the cell is sent on RNA to protein synthesis in the cytoplasm.
Two main stages:
 Transcription
 Translation

 Two main stages:


Transcription



The transfer of genetic information from DNA into an RNA molecule
Occurs in the eukaryotic cell nucleus
RNA is transcribed from a template DNA strand
Translation
 Transfer of the information in RNA into a protein.


Details:
 1. Initiation-







Promoter is the nucleotide sequence on DNA that marks where transcription of a gene begins and ends; “start” signal
Promoter serves as a specific binding site for RNA polymerase and determines which of the two strands of the DNA double helix is used as the template.
Specific nucleotide sequence at promoter is TATAAA
Called the “TATA box”; located 25-35 base pairs before the transcription start site of a gene
TATA box is able to define the direction of transcription and also indicates the DNA strand to be read
Proteins called transcription factors can bind to the TATA box and recruit
RNA polymerase; it has a regulatory function
Note: TATA box is found upstream of start site and thus is NOT transcribed by RNA polymerase

 Elongation-


RNA elongates
As RNA synthesis continues, the RNA strand peels away from its DNA template, allowing the two separated DNA strands to come back together in the region already transcribed.

 3. Termination-



RNA polymerase reaches a sequence of bases in the DNA template called a terminator.
Signals the end of the gene; at that point, the polymerase molecule detaches from the RNA molecule and the gene.
mRNA (messenger RNA) or “transcript” exits the nucleus via the nuclear pores and enter the cytoplasm

 http://wwwclass.unl.edu/biochem/gp2/m_biology/animation/gene/ge ne_a2.html



Before mRNA leaves the nucleus, it is modified or processed.
1. addition of extra nucleotides to the ends of the transcript
 Include addition of a small cap (a single G nucleotide) at one end and a long tail (a chain of 50 to 250 A’s) at the other end
 Cap and tail facilitate the export of the mRNA from the nucleus, protecting the transcript from attack by cellular enzymes, and help ribosomes bind to the mRNA
 Cap and tail are NOT translated into protein.
http://vcell.ndsu.edu/animations/mrnaprocessing/movie.htm



2. RNA splicing



Cutting-and-pasting process catalyzed by a complex of proteins and small RNA molecules, but sometime the RNA transcript itself catalyzes the process.
Introns


“intervening sequences”; internal noncoding regions
Get removed from transcript before it leaves nucleus
Exons



Coding regions; parts of a gene that are expressed as amino acids
Joined to produce an mRNA molecule with a continuous coding sequence
Cap and tail are considered parts of the first and last exons, although are not translated into proteins. http://student.ccbcmd.edu/biotutorials/protsyn/exon.html


 http://www.pbs.org/wgbh/aso/tryit/dna/protein.html
http://www.wisconline.com/objects/index_tj.asp?objID=AP1302





A typical gene consists or hundreds or thousands of nucleotides in a specific sequence, which get transcribed onto mRNA.
Translation is the conversion of nucleic acid language into polypeptide language
There are 20 different amino acids.
 A cell has a supply of amino acids in cytoplasm, either obtained by food or made from other chemicals.
Flow of information from gene to protein is based on a
triplet code: genetic instructions for the a.a. sequence of a polypeptide chain are written in DNA and mRNA as a series of three-base pairs, or codons.




To convert the codons of nucleic acids on mRNA to the amino acids of proteins, a cell employs a molecular interpreter, called transfer RNA (tRNA)
tRNA molecules are responsible for matching amino acids to the appropriate codons to form the new polypeptide.
tRNA’s unique structure enables it to be able to:


1. pick up the appropriate amino acids
2. recognize the appropriate codons in the mRNA




 tRNA is made of a single strand of RNA consisting of about
80 nucleotides
By twisting and folding upon itself, it forms several doublestranded regions in which short stretches of RNA base-pair with other stretches.
at one end of the folded molecule contains a special triplet of bases called an anticodon.


Complementary to a codon triplet on mRNA
Anticodon recognizes a particular codon triplet on mRNA
At the other end of the tRNA molecule is a site where an amino acid can attach.




Each amino acid is joined to the correct tRNA by a specific enzyme.
Each enzyme specifically binds one type of amino acid to all tRNA molecules that code for that amino acid, using a molecule of ATP as energy to drive the reaction.
The resulting amino acid-tRNA complex can furnish its amino acid to a growing polypeptide chain.

 Ribosomal RNA (rRNA)




Organelle in the cytoplasm that coordinates the functioning of mRNA and tRNA and actually makes polypeptides.
Consists of two subunits: large and small
Each ribosome has a binding site for mRNA, and three binding sites for tRNA.


E site
 Removes tRNA from ribosome
P site
 Holds the growing polypeptide
 A site
 Obtains new amino-acid-tRNA
Ribosome holds tRNA and mRNA molecules close together, allowing the amino acids carried by the tRNA molecules to be connected into a polypeptide chain.



Can be divided into same three phases: initiation, elongation, and termination.
1. Initiation
 Brings together the mRNA, a tRNA bearing the first amino acid, and the two subunits of a ribosome.
 Role is to establish exactly where translation will begin, ensuring the mRNA codons are translated into the correct sequence of amino acids.



1. Initiation (continued…)
Two steps:


1. an mRNA binds to a small ribosomal subunit. A special initiator tRNA binds to the specific codon, called the start codon, where translation begins on mRNA.
 Initiator tRNA carries the amino acid Methionine (Met); its anticodon
UAC binds to the start codon, AUG
2.A large ribosomal subunit binds to the smaller one, creating a function ribosome. The initiator tRNA fits into tRNA binding site (P site) on the ribosome. A site is vacant and ready for the next amino-acid carrying tRNA.

 2. Elongation
 Once initiation is complete, amino acids are added one by one to the first amino acid. Each addition occurs in a three step process:



1. codon recognition
 The anticodon of an incoming tRNA carrying an amino acid, pairs with the mRNA codon in the A site of the ribosome
2. peptide bond formation
 Polypeptide separates from the tRNA to which it was bound (P site) and attaches by a peptide bond to the amino acid carried by the tRNA in the A site.
 The ribosome catalyzes formation of the bond.
3. translocation




P site tRNA, moves to the E site and leaves the ribosome.
The ribosome then translocates (moves) the tRNA in the A site, with its attached polypeptide, to the P site.
Codon and anticodon remain bonded, and the mRNA and tRNA move as a unit
Movement brings into the A site the next mRNA codon to be translated, and the process begins again at step 1.

 Termination


Elongation continues until a stop codon reaches the ribosome’s
A site.
Stop codons- UAA, UAG, and UGA, do not code for amino acids but instead act as signal to stop translation.
 The completed polypeptide is released from the last tRNA and exits the ribosome, which then splits into its separate subunits.

 http://wwwclass.unl.edu/biochem/gp2/m_biology/animation/gene/ge ne_a3.html

Several ribosomes can translate an mRNA at the same time, forming what is called a polysome.



Free ribosomes


Found in cytoplasm
Synthesize proteins for use primarily within the cell
Bound ribosomes


Found on rough ER
Synthesize proteins primarily for secretion or for lysosomes




Each polypeptide coils and folds, assuming a 3-D shape, its tertiary structure.
Several polypeptides may come together, forming a protein with quaternary structure.
Overall significance:


Process whereby genes control the structures and activities of cells
The way genotypes determine phenotypes; proteins made from the original DNA nucleotides determine the appearance and capabilities of the cell and organism!



Mutation is any change in the nucleotide sequence of DNA.
Can involve large regions of a chromosome or just a single nucleotide pair, as in sickle cell disease
 In one of the two kinds of polypeptides in the hemoglobin protein, the sickle-cell individual has a single different amino acid.
 This small difference is caused by a change of a single nucleotide in the coding strand of DNA. Only ONE base pair!

 Two general categories:
 Base substitution



Also known as a point mutation
Replacement of one nucleotide with another.
Depending on how the base substitution is translated, it can result in no change in the protein (due to redundancy of genetic code), an insignficant change, or a change that significantly affects the individual.


Occasionally, it leads to an improved protein that enhances the success of the mutant organism and its descendants.
More frequently, its harmful.
o May cause changes in protein that prevent it from functionally normally.
o If stop codon is a result of mutation and protein is shortened, it may not function at all.

 Base insertions or deletions



Also known as frameshift mutation
Often has a disastrous effect
Adding or subtracting nucleotides may result in an alteration of the reading frame of the message

 all the nucleotides that are “downstream” of the insertion or deletion will be regrouped into different codons.
Result will most likely by a nonfunctional polypeptide

 What causes mutations?


Mutagenesis, or the production of mutations, can occur in a number of ways.
Errors that occur during DNA replication or recombination are called spontaneous mutations.
 Mutagen, a physical or chemical agent that causes mutations


Physical mutagen: high-energy radiation, such as X-rays and UV light
Chemical mutagen: consists of chemicals that are similar to normal DNA bases pair incorrectly.




Can also be helpful both in nature and in the laboratory.
It is because of mutations that there is such a rich diversity of genes in the living world, that make evolution by natural selection possible.
Also essential tools for geneticists.
 Whether naturally occurring or created in the laboratory, mutations create the different alleles needed for genetic research.