Protein Trafficking/Cell Organelles
What part of a protein determines where a ribosome travels to complete translation?
The N (amino) protein terminus
What is X inactivation? What is a Barr Body?
X inactivation occurs in each XX cell, wherein one X chromosome is condensed and cannot be transcribed. A Barr Body is a dense structure that the inactivated X chromosome is packaged into so it is inaccessible for transcription
What is a spliceosome?
An assembly of proteins and small nuclear non-coding RNAs. Recongizes intron exon boundaries, joining exons and splitting introns. Works with RNA binding proteins to control which sequence are spliced
What is the difference between a loss of function and gain of function mutation?
Loss of function: disrupt/loss of protein function, results from getting two non-functional copies of a gene (recessive)
Gain of function: hyperactive of an existing phenotype/increased protein function, results from only one mutant gene copy
What mutations cause sickle cell anemia and cystic fibrosis?
Sickle cell anemia: missense mutation (swapping one amino acid for another) wherein Glutamate is replaced by Valine. This causes hemoglobin misfolding
Cystic fibrosis: deletion of 3 nucleotides to lose a single amino acid (Phenylalanine deleted). This causes a mutant CFTR protein
What is endocytosis and exocytosis? How are vesicles utilized to do these processes?
Endocytosis: "within", cell absorbs molecules from extracellular environment
Exocytosis: exporting/releasing components to the outside of the cell
Vesicles fuse to the cell plasma membrane, either bringing materials in and pinching off or releasing them
Explain what happens during these two processes:
1) Chemical Modification of Histones (Histone Acylation)
2) Chromatin Remodeling
1) Acetyl groups are added by HATs (histone acetyl transferases) onto positive lysine residues. This weakens the negative charge on DNA and opens up chromatin
2) Chromatin remodeling complexes use ATP to slide nucelosomes along fibers. This increases space between nucleosomes and opens up the chromatin.
What are three events that promote mRNA stability?
5' cap modification: protects the 5' end of mRNA from degradation, necessary for nuclear export and protecting protein coding transcript
3' polyA tail: polyadenylation at the 3' untranslated region of the mRNA
RNA protein recruitment: RNA binding proteins that attach to the 3' untranslated region of DNA --> can recruit RNases or protect against RNases
What is germline transformation and how does this create genetically engineered salmon?
Germline transformation: Modify an organisms DNA when it is a zygote/early embryo (inject Cas9 donor DNA/RTV)
Result: all subsequent cells will have that modified genome
Salmon: Growth hormone gene was originally season specific, --> took constitutive enhancer from another organism and put it in the salmon
constitutive enhancer upstream from growth hormone gene, always expressing this gene now so grows faster
What is PCR? What is needed for this process?
Materials:
Template strand, primer with 3 'OH for binding, nucelotides, heat resistant DNA polymerase, heat (denatures the DNA)
Describe the Endomembrane System
Include associated organelles, their function and the topology/design
Nuclear Envelope: two membrane bilayer with pores for transport of materials and protection of nucleus
Rough ER: has ribosomes attached that make proteins
Smooth ER: no ribosomes attached makes lipids
Golgi Apparatus: protein sorting/transportation outside of cell
Lysosomes: cellular degredation
Topology: interconnected network of double membraned structures, no separation between endomembrane system components
Explain how DNA is packaged. How does this affect DNA accessibility?
DNA is wrapped around histone proteins. The DNA + histone complex is called a nucelosome. Nucelosomes assemble into chromatin
This compact storage of DNA makes it less accessible to transcribe. Specifically, heterochromatin is the highly condensed DNA, while euchromatin is more open DNA
Name and describe the three types of post translational modifications
1) Targeted destruction of proteins- ubiquitin tagged proteins are recognized by proteosomes for degredation. Utilizes E3 ubiquitin ligases to add ubiquitin to proteins. Regulates cellular protein amounts/clears up non functional proteins
2) Phosphorylation/Dephosphorylation- proteins are activated/deactivated by the covalent reversible addition of phsophate groups
3) Protein localization- conformational change of protein and transportation to the nucleus (ex: estrogen receptor)
Describe the process of AAV Gene Therapy. What is complementation?
AAV viruses transport dsDNA genome to the nucelus. The genome is not replicated or integrated into host machinery.
AAV Gene Therapy: replace AAV genome with engineered DNA and viral proteins to infect cells. The target DNA will go to the host nucleus without integration.
Result: Complementation- adding another copy of a gene to compensate for one nonfunctional copy within a cell --> helps with loss of function mutations
What are the three DNA repair systems that correct replication errors?
1) MMR: Mismatch Repair System - repair enzymes break part of the sugar/phosphate DNA background. Next new and correct nucleotides are added in and the gap is sealed --> fixes general DNA damage
2) Nucelotide Excision Repair: Enzymes detect dimers in DNA structure and cuts them out. DNA polymerase fills in the gap and ligase seals it --> fixes dimer DNA damage
3) Base Excision Repair- bad base pairings in DNA (ex: G-U) are removed. One baseless nucelotide is given the correct nucelotide pair (G-C)
Where does a protein go if it has no signaling?
Describe how ribosomes translocate to the nucleus & chloroplasts/mitochondria
Nucleus: Proteins with an internal nuclear-localization signal (NLS) are recognized. The protein is then signaled to go to the nucleus
Mitochondria/Chloroplasts: Proteins with an amino terminal signal (different than the ER signal) are recognized and translocated
No signaling: stay in cytoplasam where originally created
What is an enhancer? What is a promoter?
Explain how proteins bind to specific DNA sequences?
Promoter- general transcription factors/RNA polymerase binds to begin transcription
Enhancer- DNA elements bound by regulatory transcription factor proteins (upstream of promoter on DNA)
DNA transcription factors bind to specific enhancer sequences on the major groove of DNA. These specific pairings match in shape/chemistry and form a bond. The specific enhancer and regulatory transcription factor are bounded now.
Describe the synthesis and function of the miRNA-RISC complex
miRNA is not translated, but further processed from an miRNA coding gene
miRNA transcript is exported into the cytoplasam where it binds to itself (self complementary) and becomes double stranded. Enzymes cleave each strand of the miRNA apart and incorporate each into a protein complex
miRNA-RISC targets mRNA and base pairs with it. The mRNA is degraded/translation inhibited
Explain the process of CRISPR genome editing. How does it contribute to gene silencing/insertion?
CRISPR technology can target a specific DNA sequence by using a complementary RNA molecule to base pair and "find" the original DNA
Needed: Complementary guide RNA, Cas9 protein to guide/cut the genomic DNA of interest, donor DNA to add into the excised genomic area
Gene Silencing: Cas9 guided by RNA molecule to bad gene of interest, complementary base pairing. Cas9 cuts out this DNA (it is silenced) and the remaining ends join together (similar to deletion mutation)
Gene Insertion: Cutting the DNA and replacing with another functional piece of DNA (inserting a new gene through repair of the excision)
Describe the difference between somatic and germ cells. How does this affect there respective rates of mutation?
Somatic cells are normal tissue cells that divide regularly and become daughter cells. Germ cells are reproductive cells that are set aside early in embryonic development
Somatic cells are rapidly dividing, which means higher chance of acquiring a mutation. This mutation is heritable to daughter cells, and over many generations these mutations add up. Germ cells don't really divide much, so the chances of getting mutations are much lower
Describe how a ribosome translocates in the Endoplasmic Recticulum
What is different in the translocation of soluble vs transmembrane proteins?
A hydrophobic N-terminal signal sequence is recognized on the protein. The protein is then bound by a signal recognition particle which stops translation. The ribosome goes into the ER and translation begins again.
Transmembrane proteins with hydrophobic domains move out of the ER transportation channel and the hydrophobic part remains in the membrane
Soluble proteins are trafficked to other parts of the cell and can pass through the different membranes bc hydrophilic
Provide a summary of the steps for activating transcription
1) DNA packaged in chromatin is opened up by pioneers, HATs and chromatin remodeling complexes
2) Transcription factors bind to the specific enhancer
3) General transcription factors + RNA ploymerase bind to the promoter
4) Enhancer promoter looping (proteins bound to the promoter/enhancer bump into each other, changing the conformation of the basal transcription complex and activating RNA polymerase
Explain the process of alternative splicing and making low/high insulin affinity receptors
Alternative Splicing: produces more than one mature mRNA from the same gene
Liver cells: low affinity insulin receptors
Muscle cells: high affinity insulin receptors
Exon 11 codes for low affinity receptors
Exon 11 is present within liver mature mRNA but missing in skeletal muscle mature mRNA
In the primary (not modified) mRNA transcript exon 11 is present. The muscle cells express an RNA-binding protein that blocks exon 11 inclusion in its mature mRNA. The liver cells allows exon 11 into its mature mRNA, not expressing these RNA binding proteins. Muscle cells make high affinity receptors, liver cells make low affinity receptors
Describe the process of Retrovirus gene therapy (RVT). What is a challenge of this?
Retroviruses copy their RNA genome into a DNA genome --> this DNA copy is integrated into host cells
1) Recombiant RNA replaces viral DNA and is packaged into the virus
2) The virus targets the infected cell
3) Reverse transcriptase makes the RNA into ssDNA
4) DNA Polymerase adds nucelotides that complementary base pairs forming ds DNA
5) Integrase inserts this DNA into the host genome randomly. This adds an additional gene copy, though the mutant gene is not fixed (complementation)
Challenge: Random insertion means this new recombiant gene packaged in the virus could be placed anywhere in the genome --> could be put into coding region and disrupt functionality at other genome places
Explain the process of Strand Termination/ Dideoxy DNA sequencing
1) ddNTPs lacking a free 3' OH group are added to a mixture with dNTPs (have a free 3 'OH group), DNA polymerase, and a primer
2) ddNTPs attach to the complementary sequence and stop chain termination --> each ddNTP is put in a separate tube
3) all the DNA fragments are run on gel electrophoresis plates --> the smaller fragments move farther up the gel
4) The piece of DNA is now oriented by size and complementary base pair --> can generate the nucelotide sequence