1
Q
Background to the Hershey-Chase Experiment
A
-1800-1940s: scientists knew chromosomes were involved in genetics
The main opinion was that the hereditary part was the protein, not the NA
- Hershey and Chase wanted to solve this problem
2
Q
How does a virus infect cells (Hershey-Chase)?
A
- Inject genetic material into a cell
- Non-genetic part (protein capsid) remains outside
- Infected cells produce large amounts of the virus
- Cells burst released copied virus
3
Q
How did the Hershey-Chase experiment happen?
A
- They studied the T2 bacteriophage which infects the E. Coli bacterium
- Radioactive isotopes label the virus (sulfur on capsid, phosphorous on DNA)
- Centrifuge is used to separate T2 and E. Coli (smaller virus remained in the supernatant, infected bacteria formed a pellet)
- Deduced DNA was the genetic material
4
Q
Rosalind Franklin and Maurice Wilkins’s investigation of DNA by X-ray Diffraction
A
- X-rays directed at a material can be scattered by the material
- Works best with crystalized materials, due to repeating patterns
- DNA was arranged in such a way that it worked
- Deduced a helix structure from the images
5
Q
Watson and Crick’s model
A
- One of their first models had phosphorous on the inside
- Franklin determined bases were hydrophobic meaning they were on the inside not phosphorous.
6
Q
What is the role of nucleosomes in DNA packing?
A
- Protects DNA and allows it to be packaged
- Formed by wrapping DNA around histone proteins (octamer) allowing it to be supercoiled.
7
Q
What is the structure of a nucleosome like?
A
- Octamer has 2 copies of 4 histones (8 total)
- H1 holds DNA in place around the octamer
- ‘linker DNA’ connects nucleosomes together
- H1 binds to the 3nm fibre (Solenoid) that facilitates further packing
8
Q
What is supercoiling?
A
- DNA strands are wound around itself many times
- Aprrox. length is 2m, diameter of nucleus is 10nm
- Organizes DNA for cell division
- Controls DNA expression and its ability to transcribe or not
9
Q
What is heterochromatin?
A
Allows cell to permanently supercoil, no transcription
10
Q
What is euchromatin?
A
Promotes transcription of active chromatin
11
Q
DNA replication
A
- Helix unwinds
- H-bonds break separating 2 polynucleotide strands
- ATP moves helicase along molecules
- Separated strands become parent strands
- DNA polymerase created complimentary strands
12
Q
Helicase
A
Unwinds DNA at the replication fork
13
Q
Topoisomerase
A
Releases strand ahead of helicase
14
Q
RNA Primase
A
Primes for DNA polymerase, only one on a leading strand, many on a lagging
15
Q
DNA polymerase III
A
Links phosphate on nucleotide to 3 prime of growing strand
16
Q
DNA polymerase I
A
Replaces RNA primers with nucleotides
17
Q
DNA ligase
A
Connects gaps between Okazaki fragments
18
Q
Single Strand Binding Proteins
A
Keeps the separated strands apart so that nucleotides can bind
19
Q
Direction of Replication
A
- Initiated at many pints in Eurkaryotes
- Points are called origins of replication
- Phosphate of new DNA is added to 3’ C of deoxyribise at end of chain
20
Q
Detailed Summary of Replication
A
- Occurs during interphase, helicase unwinds DNA by breaking H bonds b/w strands
- Single stranded binding proteins keep strands apart
- DNA topoisomerase is ahead of helicase to prevent supercoiling again
- Synthesis starts on strands, continuous on leading strands, pausing on lagging (okazaki fragments)
- RNA primate first synthesizes strands
- DNA polymerase III attaches to primer, adds nucleotides
- Added through deoxynucleoside triphosphate, 2 phosphate groups released and energy released joins nucleotides in a chain
- DNA polymerase I removes RNA primers and replaces them with DNA
- DNA lipase joins okazaki fragments on the lagging
21
Q
Non-Coding Regions of DNA
A
- Areas of DNA not expressed as polypeptides but still important
- Genes are the regions of DNA that code for polypeptide, contain both intron and exon
- Introns are edited out of mRNA, translated by ribosome into polypeptide
- Therefore, only exons code for polypeptide
22
Q
What are promoters, enhancers and silencers
A
Promoters: attachment points for RNA polymerase adjacent to the gene
Enhancers: binding sites of activators, sequences that increases rate of transcription
Silencers: inhibit transcription bind to repressors
23
Q
What are Introns and Exons
A
Exons: coding regions
Introns: non-coding regions that are removed and used for other cell purposes
24
Q
What is the function of the promoter?
A
A form of non coding DNA sequence near a gene, adjacent gene is transcribed. Serves as a binding site of RNA polymerase
25
How is Gene Expression Regulated by Proteins?
- Some poteins are necessary to survival and are always expressed
- Others only need to be produced at certain times and they are regulated.
- In prokaryotes expression is regulated due to the environment
Ex. Metabolism of lactose in E. Coli
26
Impact of the environment on gene expression
- Environment of a cell affects gene expression
- Only a small number of genes determine body patterns during embryonic development
- These are regulated by morphagens that diffuse across cell surface from a concentrated source
- Regulate the rate of transcription factors resulting in activation and inhibition of genes
27
Nucleotides Regulate Transcription
- Methylation is the addition of a methyl group to DNA:
Inhibits translation by binding DNA more tightly on histones (heterochromatin)
- Acetylation is the addition of acetyl groups to histones:
Promotes transcription loosens DNA around histones (euchromatin)
28
What is the study of epigenetics
- Study of heritable changes not due to DNA
- Methylation and acetykation mark DNA with epigentic tags to affect transcription.
- Each cell has a different epigentic pattern
- Tags are erased through reprogramming when reproductive cells meet to form an embryo
29
What are the 3 stages of DNA transcription
1. Initiation:
RNA polymerase binds to promoter sequence and opens helix. Key element of promoter in eukaryotes is the TATA box (in 24% of genes)
2. Elongation:
RNA polymerase build mRNA, no primer used. Unused DNA is called the coding/sense strand. DNA that is transcribed reminds into double helix
3. Termination:
RNA polymerase stops at Termination sequence at the end of a gene. mRna dissociated from template. RNA polymerase is free to transcribe another gene
30
Post Transcription Modifications
- Only happens in eukaryotes
- Most gene expression regulation in prokaryotes occurs during transcription
- In prokaryotes transcription and translation are coupled as they have no nuclear membrane
- On eukaryites introns must be removed through splicing
31
mRNA Splicing
- Happens on the same gene in different ways
- Exons may be included or excluded
- Allows multiple proteins to be produced by a single gene varying in function
- no quality control, results in errors but is insignificant as many transcriptions occur
- errors result in a protein susceptible to degradation.
32
What is capping and tailing in splicing
Capping: adds 5' cap be transcription completes, involved in initiation
Tailing: poly-A tail added after transcriptionnis completed, prevents degradation of mRNA
33
Ribosomes
- composed of protein (stability) and RNA (catalysis)
- composed of a small (mRNA binding) and large (tRNA binding) subunits
- If protein is for intracellular use, ribosome is free in cytoplasm
- If protein is for secretion ribosome binds to ER
34
Determination of ribosome location
- bound or free is determined by the presence of an initial signal sequence
- signal sequence attaches to a signal recognition protein (SRP) which stops translation until binding to ER
- After binding translation restarts
- New protein is transported to the golgi or lysosome
- Signal sequence removed an SRP released
35
tRNA
- transfers AA from cytoplasm to a polypeptide
- gathers AA's when activated by a specific enzyme, requires ATP
- 20 enzymes specific for 20 AAs and tRNAs
1. Enzyme binds ATP to AA to form AA-AMP (diphosphate released, link via high E bond)
2. AA coupled to tRNA and AA released
- AA is covalently bonded to tRNA
36
3 stages of translation
1. Initiation - Ribosome units and tRNA assemble at mRNA
2. Elongation - elongation of polypeptide chain
3. Termination - completed polypeptide released ribosome complex disassembles
37
Initiation in translation
1. mRNA binds to the small subunit of a ribosome
2. Small subunit moves along the mRNA in a 5' to 3' direction until it reaches the start codon (AUG)
3. tRNA carrying methionine binds to AUG
4. A large subunit of ribosome binds to the tRNA (p site) and the small subunit to form the complex.
38
Elongation in translation
1. A second tRNA pairs with the next codon in the A site
2. AA carried by the tRNA in the P site is covalently attached by a peptide bond to the AA in site A
3. tRNA in P site is now deacylated (no AA) while the tRNA in site A carries the peptide chain
4. Translocation: ribosome moves one codon along the mRNA
5. Another tRNA binds to A site
6. ELongation repeats until a stop codon is reached
39
Termination in translation
1. when a stop codon is reached translation stops:
- polypeptide chain released
- ribosome complex disassembles
40
Coupling of transcription and translation in prokaryotes
Prokaryotes: Ribosomes can be adjacent to the chromosomes as there is no nucleus, translation happens immediately after transcription
Eukaryotes: mRNA needs to be spliced then relocated from nucleus to cytoplasm, thus it can't be coupled
41
Polysomes
Structure that consists of multiple ribosomes attached to a single mRNA enabling the cell to quickly create many copies of the required polypeptide.
42
Primary structure
order of AA in the polypeptide chain, determines all levels because it determines how R groups of AAs will interact
43
Secondary structure
Chain of AAs has polar covalent bonds within its backbone. Folds so that H-bonds are between carboxyl groups and amine of another chain. Can be a-helix or b-pleated sheet or just a random coil.
44
Tertiary structure
Overall 3D structure from different interactions of R groups:
Ionic bond: +ve R with -ve R
Hydrophobic: AA drawn into the center
Hydrogen bond: polar R with polar R
Disulphite bridge: R group of cysteine can form covalent bond with another
45
Quarternary structure
More than one polypeptide chain fitted together. Many or prosthetic groups
- Prosthetic group: inorganic compound in a structure
- Conjugated protein: protein containing a prosthetic
