DNA Study Guide

Made by Shane Tang ©

History of DNA

• Frederick Griffith
o His goal was to develop a vaccine against pneumonia
o He found a smooth coat strain that kills the mouse and a rough coat strain
that did not hurt the mouse.
 Found that heat could inactivate the smooth coat
o But the inactivated smooth coat plus the rough coat caused death
o There was something in the smooth strain that transformed the rough
strain into a lethal form
• MacLeod, Avery, and MacCarthy
o Extended Griffith’s work
o Figured out that the change in the rough strain into the lethal strain was
caused by DNA
 First used process of elimination: DNA, RNA, and protein
o They used DNAse and the mouse lived
 When using protease and RNAse, the mouse died
 This explains that the DNA is causing the change
• Hershey and Chase
o Figured out that DNA is the genetic material
o “Blender experiment”
 DNA is rich in phosphorus not in protein
 Proteins are rich in sulfur, not much in DNA
 Two bacterium infect sulfur and phosphorus
• One bacteriophage’s DNA is incorporated with phosphorus
• The other bacteriophage’s protein coat is incorporated with
sulfur
 Put them both in a blender so it breaks the bacterial cells away
from any viral material remaining outside them
 Protein did not enter the bacteria but the DNA had
 Radioactive DNA had been passed down to these cells offspring
but not the radioactive proteins.
• Meselson and Stahl (1958)
o Determined that DNA replication was semi-conservative
 Semi-conservative means when DNA was replicated, each of the
two double-stranded DNA helices consisted of one strand from the
original helix and one newly synthesized strand
 The other two types of DNA replication that is not found to be
biologically significant are conservative and dispersive
• Conservative – the original strand does not split and others
are created purely from the newly synthesized
 • Dispersive – parts from the original strand mixes with the
newly synthesized strand
• Erwin Chargaff
o Chargaff’s Rule
 States that DNA from any cell of all organisms should have a 1:1
ratio of pyrimidine and purine bases and, more specifically, that the
amount of guanine is equal to cytosine and the amount of adenine
is equal to thymine
 Purines are adenine and guanine, they have two rings
 Pyrimidines are thymine and cytosine, they have three rings
• James Watson and Francis Crick
o Determined that the structure of DNA was a double-helix
 DNA consists of…
• Phosphate Group
• Deoxyribose (Pentose Sugar)
• Nitrogenous base
 Guanine and Cytosine have three hydrogen bonds in between
 Adenine and Thymine have two hydrogen bonds in between
• Rosalind Franklin
o Watson and Crick ripped info from her
o She, with Maurice Wilkins, used X-ray diffraction (crystallography) to
look at the shape and structure of DNA
 She found that phosphate was attached to sugar in a continuous
chain
• Arthur Kornberg
o Isolated DNA polymerase 1 from E. Coli
o Found everything needed for DNA replication
 All four dNTP’s (nucleotides triphosphates)
 Mg2+ (Magnesium)
 Fragment of DNA
 DNA polymerase 1
• George Beadle and Edward Tatum (1933)
o One gene can equal one enzyme
o One gene can equal one protein
o One gene can equal one polypeptide
 Now scientists have figured out that one gene can equal several
proteins
• Thomas Hunt Morgan
o Fruit fly (Drosophila Melanogaster) research
o Developed the chromosome theory
 Genes are carried on chromosomes and are the mechanical basis of
heredity
DNA Replication

• DNA has three types: A, B, and Z

• The things that make it up are in the top section
• Phosphodiester bonds join the nucleotides, creating the sugar-phosphate backbone
• The 5’ end is the phosphate while the 3’ end is the hydroxide
• The two strands are connected by hydrogen bonds between the nitrogenous base
pairs
• The two strands are anti-parallel because of the 5’ and the 3’
• Diagram

• The leading strand is unbroken while the lagging strand is broken because it
replicates in a 5’ to a 3’ manner
• List of helpers in DNA Replication
o Helicase – breaks apart the strands by finding an adenine-thymine rich
area because their bonds are easier to break because they only have two
hydrogen bonds instead of the three
 The initiation point is called the replication fork or the origin of
replication
o SSBs (Single Strand Binding Proteins) – keep the strands from joining
back together after helicase breaks them apart
o RNA primase – synthesizes the first nucleotides of the new strand
o DNA polymerase – builds the new strand of DNA (needs RNA primase
because polymerase can’t build from scratch)
 o Exonuclease (a different kind of DNA polymerase) – replaces the RNA
primer with DNA
o Ligase – joins the Okazaki fragments (sections of DNA) together on the
lagging strand
o Gyrase (or also Topoisomerase) – reduces supercoiling so DNA does not
break
• When DNA polymerase connects with the parent strand, it has three triphosphate
nucleotides but two are broken off because energy is released
o Energy used to polymerize the new strand of DNA
• Difference between mitochondrial DNA and nucleus DNA
o Unlike nuclear DNA, whose genes are rearranged in the process of
recombination, there is usually no change in mtDNA from parent to
offspring. Because of this, and the fact that the mutation rate of mtDNA is
higher than that of nuclear DNA and is easily measured, mtDNA is a
powerful tool for tracking matrilineage, and has been used in this role for
tracking the ancestry of many species back hundreds of generations.
Human mtDNA can also be used to identify individuals
o Mitochondrial DNA is also circular and has no introns
• Introns and Exons
o Introns are DNA regions in a gene that are not translated into a protein
 These are present in pre-mRNA and removed by a process called
splicing during the processing to mature RNA
 After the splicing, the mRNA consists only of exons, which are
translated into a protein
o Exons are nucleic acid sequences that are represented in the mature form
of an RNA molecule after a) portions of a precursor RNA, introns, have
been removed by cis-splicing or b) two or more precursor RNA molecules
have been ligated by trans-splicing.

Transcription

• Transcription is the process wherein a molecule of mRNA is synthesized along a

template strand of DNA
• It occurs in the nucleus of eukaryotic cells, the nucleolus to be accurate
• RNA
o Single-strand
o Uses the sugar ribose instead of deoxy-ribose
o Uses the base uracil instead of thymine
o Working copy, leaves the nucleus to make proteins
• In transcription, the RNA builds in a 5’ to 3’ manner
o The template strand is 3’ to 5’ while the informational strand is 5’ to 3’
• Helpers in transcription
 o Promoter site – they are recognized by a factor called SIGMA. It is
SIGMA’s job to tell the DNA dependent RNA polymerase where to begin
transcription
 In eukaryotes, a TATA box is typically present in the promoter,
consisting of a nucleotide sequence like “TATAAAA”
• Aids in the recognition of the promoter
o Transcription factors – proteins that locate and bind to the TATA box.
They also help RNA polymerase recognize and bind to the promoter
o RNA polymerase 2 – responsible for transcribing the pre-mRNA strand
• Initiation – Part 1
o Transcription factors locate TATA box which is a place of promoters
o RNA polymerase 2 comes, aided with transcription factors, locates the
start point and begins to unwind the DNA helix
• Elongation – Part 2
o RNA polymerase 2 assembles the RNA nucleotides that complement the
template strand
o As RNA polymerase 2 continues along the transcription unit, the newly
synthesized RNA strand separates from the template
• Termination – Part 3
o As RNA encounters the end, the enzyme encounters a terminator, or a
sequence of nucleotides telling it to stop
o Transcribes the terminator, then transcribes 10-15 more nucleotides before
getting released
o It then breaks off, and the DNA goes back to the double helix form
• RNA processing
o This is needed after transcription to get the mRNA ready for the cytoplasm
 The cytoplasm is where the mRNA goes after transcription
o On the 5’ end, a methyl-guanosine cap is added
 Protects the mRNA from degradation by hydrolytic enzymes as it
travels through the cytoplasm; it also serves as a signal for the
attachment of the ribosome
o On the 3’ end, a poly-A tail is added
 Made from 100-200 adenine nucleotides
 Protects the mRNA from degradation and helps in recognition by
the ribosome
o Then cut out the introns so only the exons are left, method is called
splicing
Translation

• Translation occurs in the ribosome

• Polypeptide is a chain of amino acids, ex. Leu
• rRNA (ribosomal RNA) – ribonucleic acid which, together with proteins,
comprises the ribosome, the physical site of translation
• Initiation – Part 1
o mRNA transcript comes together with the smaller of the two ribosomal
subunits and a tRNA molecule
 tRNA (transfer) carries the 1st amino acid of the polypeptide
o Puts it on the start codon, AUG
o The second and larger ribosomal subunit arrives, marking the end
 Contains the P, and A sites
• A site contains the incoming tRNA
• P site holds the tRNA that carries the growing Polypeptide
strand
• Elongation – Part 2
o Codon recognition
 Incoming tRNA contains the anti-codon, while the strand already
has the codon on it.
• Codon is a set of three nucleotides
 Codons and anti-codons are held together by hydrogen bonds
o Bond formation
 The large ribosomal subunit acts as a ribozyme, joins the two
amino acids together with a peptide bond
 o Translocation
 tRNAs move down together one site with the mRNA template
• Termination – Part 3
o Terminated when a stop codon is reached in a mRNA transcript
o Release factor – protein that recognizes and binds to the stop codon
 Adds a water molecule and that releases the polypeptide
o Once polypeptide is released, the ribosomal subunits and other factors
break it apart