Notes 10/28

3. Steps of DNA replication (DRAW PICTURES!) bacteria replication
a. Ori C: Where DNA replication begins on the bacterial chromosome
i. Bacteria chromosome is circular and before division is supercoiled on itself
ii. First bacteria must be unwound/uncoiled and unzipped somewhere on the DNA but cannot uncoil the entire chromosome; only a small part of it – locally
1. Only a teeny tiny bit is going to unwind the rest will stay bunched and coiled up on top of itself
iii. This unwinding and unzipping on part of the DNA is at the oriC site
iv. Ori C → origin of chromosome replication; where the replication of the chromosome is going to start
1. Has a lot of As & Ts little Gs & Cs at it because this is the sequence of bases that is the easiest to unzip because they only have a double hydrogen bond instead of a triple hydrogen bond
2. Enzymes bind to the DNA here
a. DNA A → not made out of DNA it is a protein enzyme that binds to oriC site on DNA – recruits helicase which unwinds and unzips our DNA in that tiny local section
i. Brings in another protein DNA B
b. DNA B helicase → enzyme that uncoils the DNA supercoils
b. DNA B Helicase uncoils supercoils around oriC
i. Unzips DNA double strands
ii. Primase → enzyme that synthesizes a small swatch of RNA that is complementary to part of the oriC sequence/DNA sequence – which allows DNA polymerase to bind to DNA strand, and start copying it
1. Primease, single-stranded binding protein, attaches to unzipped DNA, synthesizes RNA primer to prevent re-annealing
2. Primase synthesizes a little primer swatch of RNA complementary to DNA at oriC
c. DNA polymerase attaches to the RNA primer, synthesizes new strand of DNA that is complementary to the template strand, moving in 5’ → 3’ direction
i. DNA polymerase enzyme is going to bring in bases that are complementary to the bases on the single strand of DNA (template strand) and match them up and hook the bases together using a sugar phosphate backbone
ii. This enzyme can only move/travel in a 5’ → 3’ direction
1. This is a problem because DNA is antiparallel
d. One strand is synthesized continuously, but opposite strand has to be synthesized in little portions – resulting fragments are called, Okazaki Fragments, on lagging strand
i. These fragments are going to have to be patched together – this is done by the enzyme DNA Ligase – all it does is patch together Okazaki Fragments
ii. Since the chromosome is circular and you are going around in opposite directions – eventually you will reach the other side of the circle and DNA replication would be finished
e. DNA replication enzymes are moving in opposite directions around the circular chromosome
i. Go around in opposite directions and meet at the opposite side, terminus site, which is where DNA replication is going to stop.
ii. You end up with two interlocking circles which is an issue because we want each chromosome to go off onto their own
f. Interlocked circles from the complete DNA replication have to be uninterlocked in order for the replication to be completely finished 8 → o o
i. Done by DNA gyrase and topoisomerase IV

Background on Lab
Phenotype – Manifestation of the genes – set of genes that are expressed – doesn’t change in sunlight
- Observed characteristics
Genotype – The genes that a person has – blue eyes, pale, skin, brown hair, etc. – can change in the sun
- Many bacteria have genes that are not always expressed – not always on – only expressed under certain conditions.
- Quorum Sensing – Ability of some bacteria to change phenotype at different population densities.
o Special example of genes that can only be shown under certain circumstances.
o Ex. Suicide at high population densities
• Ex. Streptococcus pneumoniae - genes for signaling molecule and receptor are ALWAYS on, when all the receptors have the signal molecule bound to them another gene, Lyt A, which kills the bacteria from the inside out
o Ex. Creates a biofilm in high populations – biofilm made out of rhamnolipids (carb/lipid complex) and pyocyanin (protein)
• Ex. Pseudomonas aeruginosa – signal molecule is a chemical called homoserine lactone (HSL) – when all of the receptors have molecule attached to them the genes that are expressed are the rhamnolipids genes and the pyocyanin genes – which allows our bacteria to make the elaborate biofilm. The HSL gene and receptors are ALWAYS on
• The biofilm allows the bacteria to stick to surfaces and not be washed away – and allows the bacteria to not be phagocytosed by things that are bigger than the bacteria
• At some point there will be some isolated bacteria that can’t communicate with the rest very well and they will break out of the biofilm and go create their own somewhere else
o Ex. Expresses a protein, luciferase, at high populations – which emits a very faint blue/green light
• Ex. Vibrio fischeri – gram-negative bacteria water bacteria that live in salt water, ocean. When all the receptors have signal molecule bound to them, gene for luciferase is expressed. Makes the bacteria glow a blue/green color
• Luciferase – light bringing enzyme
• Uses same signaling molecule as pseudomonas aeruginosa
• Lives in the ocean, so they will never be at densities that are high enough to make these bacteria glow blue/green
• Vibrio fischeri has a symbiotic (doesn’t cause harm to either one, both work together) relationship squid: eurypmna scolopes
• Eurypmna scolopes – nocturnal, predator to some small animals, and prey to other larger animals – has a special organ in its head, the light organ, which is specifically colonized with the bacteria that give off blue/green light. Allowing a very high population density to be in the head of the squid (small space) causing the squid to glow
• Bacteria get into the head of the squid from the surrounding ocean
• The moon shining down on the squid causes it to cast a shadow allowing the prey and predators to see the squid due to the shadow – the blue/green light is about the color of moon light under 10 ft. of sea water causes the shadow to be obscured because it glows canceling it’s own shadow
• In lab there was a cultural of Vibrio fischeri – and hoping that if we go into a very dark room, bacteria will emit a faint blue/green light (color of moonlight at 10-15 ft. of sea water)

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License