Nucleic Acids Flashcards
Background to the Hershey-Chase Experiment
-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
How does a virus infect cells (Hershey-Chase)?
- 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
How did the Hershey-Chase experiment happen?
- 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
Rosalind Franklin and Maurice Wilkins’s investigation of DNA by X-ray Diffraction
- 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
Watson and Crick’s model
- One of their first models had phosphorous on the inside
- Franklin determined bases were hydrophobic meaning they were on the inside not phosphorous.
What is the role of nucleosomes in DNA packing?
- Protects DNA and allows it to be packaged
- Formed by wrapping DNA around histone proteins (octamer) allowing it to be supercoiled.
What is the structure of a nucleosome like?
- 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
What is supercoiling?
- 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
What is heterochromatin?
Allows cell to permanently supercoil, no transcription
What is euchromatin?
Promotes transcription of active chromatin
DNA replication
- Helix unwinds
- H-bonds break separating 2 polynucleotide strands
- ATP moves helicase along molecules
- Separated strands become parent strands
- DNA polymerase created complimentary strands
Helicase
Unwinds DNA at the replication fork
Topoisomerase
Releases strand ahead of helicase
RNA Primase
Primes for DNA polymerase, only one on a leading strand, many on a lagging
DNA polymerase III
Links phosphate on nucleotide to 3 prime of growing strand
DNA polymerase I
Replaces RNA primers with nucleotides
DNA ligase
Connects gaps between Okazaki fragments
Single Strand Binding Proteins
Keeps the separated strands apart so that nucleotides can bind
Direction of Replication
- 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
Detailed Summary of Replication
- 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
Non-Coding Regions of DNA
- 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
What are promoters, enhancers and silencers
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
What are Introns and Exons
Exons: coding regions
Introns: non-coding regions that are removed and used for other cell purposes
What is the function of the promoter?
A form of non coding DNA sequence near a gene, adjacent gene is transcribed. Serves as a binding site of RNA polymerase
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
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
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)
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
What are the 3 stages of DNA transcription
- Initiation:
RNA polymerase binds to promoter sequence and opens helix. Key element of promoter in eukaryotes is the TATA box (in 24% of genes) - Elongation:
RNA polymerase build mRNA, no primer used. Unused DNA is called the coding/sense strand. DNA that is transcribed reminds into double helix - 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
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
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.
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
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
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
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
3 stages of translation
- Initiation - Ribosome units and tRNA assemble at mRNA
- Elongation - elongation of polypeptide chain
- Termination - completed polypeptide released ribosome complex disassembles
Initiation in translation
- mRNA binds to the small subunit of a ribosome
- Small subunit moves along the mRNA in a 5’ to 3’ direction until it reaches the start codon (AUG)
- tRNA carrying methionine binds to AUG
- A large subunit of ribosome binds to the tRNA (p site) and the small subunit to form the complex.
Elongation in translation
- A second tRNA pairs with the next codon in the A site
- AA carried by the tRNA in the P site is covalently attached by a peptide bond to the AA in site A
- tRNA in P site is now deacylated (no AA) while the tRNA in site A carries the peptide chain
- Translocation: ribosome moves one codon along the mRNA
- Another tRNA binds to A site
- ELongation repeats until a stop codon is reached
Termination in translation
- when a stop codon is reached translation stops:
- polypeptide chain released
- ribosome complex disassembles
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
Polysomes
Structure that consists of multiple ribosomes attached to a single mRNA enabling the cell to quickly create many copies of the required polypeptide.
Primary structure
order of AA in the polypeptide chain, determines all levels because it determines how R groups of AAs will interact
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.
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
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
