Chapter 9
DNA and its role in heredity
The discovery of the three dimensional shape of DNA was a milestone in biology
A variety of evidence was used to determine the shape of DNA around the middle of the 20th century
This allowed us to determine how DNA replicated and how it directs the synthesis of proteins
Chargaff
DNA composition studies concluded that there were four nitrogen bases (adenine, thymine, guanine and cytosine)
Chargaff found that the amount of each base varies from species to species
Also found that amount of each base isn’t equal in each species
They are present in characteristic ratios.
Chargaff Rules
If a species is 19% Guanine, what percentages represent the other 3 bases?
If a species is 35% adenine, what percentages represent the other 3 bases?
The race for the structure of DNA
From 1950 – 1953 there was an unofficial competition between Cal Tech (Linus Pauling) and Cambridge (James Watson and Francis Crick).
Pauling (already famous for his work with chemistry) would be the first to publish a theoretical structure…but it was significantly flawed.
Franklin and Wilkins
· Rosalind Franklin worked in Wilkins lab and both of them had made significant strides in the structure of DNA.
· Franklin was an X-ray crystallographer.
· It was from one of her x-rays (#51) that she was able to tell that the structure was a double helix. That picture and who was able to see it would prove to be significant.
· Franklin took many pictures of DNA (really it’s a shadow cast by x-rays passing through the molecule) both dehydrated and in an aqueous solution.
· #51 showed a clear helical pattern … to people who know this stuff…I see a fuzzy X—but that’s just me.
Watson and Crick
· James Watson – American biologist
· Francis Crick – English chemist
· Saw picture #51 and combined it with Chargaff’s rules to elucidate the structure of DNA.
· Determined the structure was a double helix
· Published their results in April 1953 issue of Nature
· Nobel Prize – 1962: Watson, Crick and Wilkins shared the 1962 Nobel Prize for the structure of DNA. Franklin would have been included but she died of ovarian cancer before the Nobel Prize was awarded.
The Structure of DNA
DNA uses nucleotides as its building block.
Nucleotides consist of phosphate, sugar and a base
Nitrogen Bases
· There are 4 nitrogen bases in DNA: Adenine, Thymine, Guanine and Cytosine.
· A and G are purines which are lager (double rings)
· T and C are pyrimidines which are smaller (single rings)
· A always bonds with T and C always bonds with G
Antiparallel strands
· Phosphate to sugar bond involves carbons in the 3, and 5’ positions.
· DNA molecule has a “direction.”
· Complementary strand runs in opposite direction
The 2 strands of DNA lead us to replication…
Watson and Crick in their 1953 publication in Nature said the following:
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
DNA Replication
There were three alternative models of DNA replication:
Semi Conservative
Through experimentation we discovered that DNA replicates semiconservatively. Which means each strand of the parental DNA acts as a template for a new strand, which is added by base pairing.
DNA replication occurs in 3 basic steps
Starting point The parent molecule has 2 complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner. A with T and C with G
DNA Replication – An overview Step 1
The DNA strands separate.
DNA Replication – An overview Step 2
Each parental strand now serves as a template that determines the order of nucleotides along a new complementary strand
DNA Replication – An overview Step 3
The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.
Replication is fast and accurate
A human body cell contains ~ 6 billion bases and is replicated in ~ 8 hours…that’s ~ 16.6 million bases per minute or ~ 275,000 bases per second
All with amazing accuracy…about one error per 1 billion bases.
Replication Forks and Bubbles
Replication begins at locations called “origins of replication”
A bacterial cell has only one origin of replication but eukaryotes have many origins of replication. ”
DNA Polymerase
Once the DNA strand has been opened up, an enzyme group of enzymes called DNA polymerases cause complementary bases to be attached (called elongation of the new DNA strand)
Each of the new nucleotides are actually triphosphates that loses two of its phosphate groups and that provides the energy of polymerization (attachment).
DNA polymerase is the enzyme that lines the triphosphate up and catalyzes this reaction.
Triphosphates
Deoxyribonucleoside triphosphates (dNTP’s)
That’s a mouthful…they lose two of their phosphates when they attach.
That gives them the energy to attach.
Elongation of a New DNA Strand
Here’s where that 3’ and 5’ stuff is important.
Look at the picture below and notice which side has an exposed hydroxyl group.
Direction of replication
Since only the 3’ end of the new strand can accept new nucleotides, only the 3’ to 5’ side of the parental DNA can accept nucleotides on its complementary strand in the direction of replication (towards the fork)
What about the other side?
Since only the 3’ end of the new strand can accept new nucleotides, the parental strand that is 5’ to 3’ gets a complementary strand added in fragments moving away from the replication fork. These fragments are called Okazaki fragments
Leading and Lagging
The side that gets to go in the direction of replication (towards the fork) moves faster and is called the leading strand.
The side that adds new nucleotides in Okazaki fragments moves slower and is called the lagging strand.
Primers
DNA polymerase cannot initiate synthesis of a new strand of nucleotides…they can only add nucleotides to the 3’ end of an already existing chain that is base-paired with the template strand.
Therefore, a primer made of about 10-12 RNA nucleotides is used to start a new chain.
Primase is the enzyme the puts the RNA primer in place.
Later a DNA polymerase will replace the RNA with DNA
Enzymes and other helpful proteins
· Helicase – Unwinds the DNA
· Single Strand Binding Protein – Hold DNA strands apart
· Primase – puts down RNA primer
· Ligase – Joins Okazaki fragments together
· DNA polymerases
o Adding new nucleotides to 3’ end (elongation)
o Replacement of RNA primer with DNA
·
Proofreading
The mistakes made in replication are fixed in a number of ways:
- DNA polymerase will check it when it adds it and will correct it immediately.
- Nucleotide excision repair – when mistakes evade proofreading. A cell will use a variety of enzymes for mismatch repair. (nuclease cuts out incorrect base and correct one is filled in by DNA polymerase and fixed by ligase.
Over time, we lose a little DNA
Because DNA can’t attach nucleotides to the 5’ end of the daughter strand , the RNA primer on the very end of the lagging strand never gets replaced by DNA.
Thus the lagging strand becomes shorter each time replication occurs
My telomeres are shorter than yours…and that depresses me
We have “spare DNA” at the very end of each of our chromosomes
This spare DNA is called a telomere and is made of “junk DNA –no genes, just repeating TTAGGG…you have between 100 and 1000 repetitions.
So what’s the significance of telomeres
We can make more telomeres using telomerase which is only available in some cells (such as reproductive cells)
Without telomerase, the telomeres eventually run out which scientists believe is a key factor in aging and death
Scientists have found telomerase in some cancerous cells.
From Gene to Protein - Chapter 10
What we know already
• We know DNA is responsible for phenotype.
• But how is DNA responsible? It codes for proteins responsible for phenotype.
• Example: Pea plants can be tall or short. Short pea plants don’t make gibberellin which is a plant growth hormone. They lack the enzyme for gibberellin synthesis. The recipe for that enzyme is lacking in their DNA
Connection between Genes and Proteins
• 1909 - Garrod first suggested this connection with what he called “inborn errors in metabolism.”
• Alkaptonuria – urine contains dark chemical alkapton because body lacks enzyme to break down alkapton. ( page 188)
• He theorized that this lack of enzymes was inherited and termed it an “Inborn error of metabolism”
• Apply the Concept page 189
One gene – one polypeptide
• First hypothesis: One gene gives rise to one enzyme.
• However, since not all proteins are enzymes, more accurately: one gene gives rise to one protein.
• However, since some proteins are made of more than one polypeptide chain the most current and accurate way to say this is one gene gives rise to one polypeptide.
• It’s termed the one gene-one polypeptide hypothesis and is currently being shot down because of exceptions. It’s a good general rule though.
How DNA becomes a protein
DNA is transcribed and translated to make a protein. The general steps involved are collectively called the “Central Dogma of Molecular Biology”
DNA vs RNA
4 kinds of RNA
• Messenger RNA (mRNA) – DNA is copied into mRNA during transcription.
•
• Transfer RNA (tRNA) used during translation to carry the correct amino acid.
• Ribosomal RNA (rRNA) – folded strands of RNA used to make a ribosome. Site of protein synthesis.
• Small Nuclear RNA(snRNA)- Combined with proteins to make a structure called a spliceosome which helps with editing the RNA molecule.
Protein Synthesis varies between prokaryotes and eukaryotes
• Prokaryotes have no nucleus so the 2 steps, transcription and translation, both occur in the cytoplasm
• Eukaryotes have 3 steps, transcription and RNA processing occurs in the nucleus, and translation occurs in the cytoplasm
From DNA to Protein
• Remember that our ultimate goal is a protein. We’ll cover the three steps (transcription, processing and translation) in detail later.
• But, for now, how does mRNA code for proteins?
– There are 20 amino acids. How do you code for 20 amino acids with only 4 nucleotide bases?
– In order to get at least 20 different combinations, we have to use at least 3 nucleotides.
– Codons are blocks of 3 mRNA nucleotides that code for an amino acid.
Breaking the Genetic code
• scientists have determined which codons code for which amino acids.
Nirenberg and Matthei determined that the 1st codon – amino acid match: UUU codes for phenylalanine
• They did this by taking a chain of uracil and adding it to a test tube of ribosomes and nucleotides…they got a chain of phenylalanine
• The genetic code
•
Chains of mRNA come about during transcription as a copy of the DNA is made.
DNA = TTAGACTAG mRNA = AAUCUGAUC
• In that chain of mRNA there are 3 codons: AAU, CUG and AUC
• Each codon can be matched up to an amino acid.
The genetic code
• Same for all life…called universal for that reason.
• Code is one of the strongest supporting arguments for a common origin for all life
• Code is redundant but not ambiguous.
• A reading frame is a set of codons read correctly. The reading frame can be shifted and the results are almost always devastating.
Practice
• AAU CUG CCC codes for which amino acids?
• AUG UUU UUA codes for which amino acids?
Transcription
http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/transcription.swf
RNA processing
• Before leaving the nucleus, the newly formed mRNA strand is modified by:
• Adding a 5’ cap
• Adding a poly A tail on the 3’ end
• Cutting out the introns
• Splicing exons together
• Alteration of mRNA ends
• A modified guanine is added to the 5’ end. It will help protect the molecule from degradation and it will signal the attachment to a ribosome in the cytoplasm
• A series of 50 to 200 adenines are added to the 3’ end. This is called a poly A tail. Helps mRNA to leave the nucleus, prevents degradation and signal attachment of a ribosome
Exons and Introns
• Sections of DNA that code for a protein will include interspersed sections that interrupt. In mRNA they are removed. These are called “intervening sequences” or introns. (Also called “junk DNA”)
• When introns are cut out the remaining mRNA is eventually expressed and are called exons.
mRNA splicing
• Introns are cut out and exons are pasted together.
• snRNA forms a complex with other proteins to make a spliceosome which recognizes introns and cuts them out.
Translation: a closer look
• The key RNA in translation is transfer RNA.
•
tRNA is a chain of about 80 or so bases. Three of bases are called the anticodon.
The anticodons are complementary to the codon in mRNA.
The top of the structure has an amino acid.
• How the amino acid joins
• The amino acids (remember that there’s 20 of them) are in abundance in the cytoplasm.