Term
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Definition
DNA RNA Protein
Retrovirus |
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Term
| Transcription vs. Replication |
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Definition
Transcription is similar to DNA replication
Makes a new complementary strand, but made of ribonucleotides
RNA pol does not need a primer (de novo)
Multiple RNA pol can work on the same strand
RNA pol has lower fidelity
o Important difference
Replication of the whole genome once
Transcription of a part of the genome multiple times |
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Term
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Definition
| mRNA (messenger RNA-codes for protein), rRNA (ribosomal RNA), tRNA (transfer RNA), snRNA (small nuclear RNA, splicing), snoRNA (used to process/modify rRNA) |
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Term
| Eukaryotic vs. Prokaryotic - Gene |
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Definition
Euk
• TATA Box
• Introns
• Monocistronic
Prok
• -35 Box, -10 Box
• TATA Box
Polycistronic |
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Term
| Eukaryotic vs. Prokaryotic - Transcriptional Regulation |
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Definition
Euk
• more refined, development
• Differentiation = making a cell specialized
• Depends on the control of genes
Prok
• main goal is to quickly change depending on the surrounding, Environmental changes (oxygen, food, temperature) |
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Term
| Eukaryotic vs. Prokaryotic - Transcription |
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Definition
Euk
• Has nuclear envelope
• Transcription separate from translation
• Pre-mRNA must be processed to mRNA
• Alternative splicing
• Monocistronic
Prok
• No nuclei
• Coupled transcription and translation
• Polycistronic |
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Term
| Eukaryotic vs. Prokaryotic - Transcription Initiation |
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Definition
Euk
• General transcription factors (GTF)
o Finds the promoter
o Closedàopen complex
• All GTFs + RNA pol = pre-initiation complex
• TATA box is distorted by insertion of a β-sheet
Prok
• σ factor
o Finds the promoter
o Closedàopen complex |
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Term
| Eukaryotic vs. Prokaryotic - Promoters |
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Definition
Euk
• Four different elements
o TATA Box
o BRE
o INR
o DPE
• TATA box
o Helps locate the start site
o Change in efficiency
• TATA-less have GC-boxes
o House-keeping genes
Prok
• UP element, -10 and -35 boxes, extended -10, Discriminator |
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Term
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Definition
• Protein bound to DNA protects it from digestion by DNase I
• Used to find the site in DNA which the protein binds to
• The DNA is radiolabeled (32P)
• Binding affinity can be estimated by protein concentration |
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Term
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Definition
• Electrophoretic Mobility Shift Assay (gel shift assay)
• Used to find where the protein binds specifically
• In a gel, protein-DNA complex is slower than free DNA (Shift)
o Found ‘higher’ in the nondenaturing gel
• Can be used in conjunction to mutagenesis
• DNA is radiolabeled for identification
• Addition of antibody (Super-shift)
• For specificity or disruption of DNA-protein interaction |
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Term
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Definition
• A known protein is used as ‘bait’
• Hybridized with DNA Binding Domain (DBD)
• Found in the pGILDA vector
• Screened against a library, the ‘prey’ proteins
o Screening
You start with a single known protein (bait)
Screen against a million of possible interactors
Potential interactors can grow in restrictive media
These are then ‘patched’ in a second isolation step
Blue colonies in galactose only have their plasmids removed
Plasmids are then sequenced
Y2HS must be followed by co-localization and co-IP
• Hybridized with Activating Domain (AD)
• Found in the pJG4-5 vector
• If the bait-prey interact, the DBD-AD are close enough
• Leads to activation of the reporter gene
• Can be seen, such as blue colonies |
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Term
| RNA pol in Eukaryotes is made up of many different subunits |
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Definition
o (2) Alpha subunits: assist in enzyme assembly, promoter recognition
o (2) Beta subunits: catalytic center, Mg++ binging on center cleft, resemble pincer in claw shape model
o Omega Subunit: restore denatured RNA polymerase in vitro to its fully functional form |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
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Definition
| responsible for promoter recognition in prok |
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Term
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Definition
o Polyclonal antibody may disrupt protein-protein interaction
o Monoclonal antibody is good for endogenous co-IP
o Epitope tag is added to protein of interest
o Has an antibody against it that can be used for co-IP |
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Term
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Definition
o Upstream activation sequence (UAS)
Increase expression, upstream, near
o Enhancers
Increase expression, close or far, up or downstream
o Silencers & repressors
Decrease expression, near or far, interfere with TF
o Boundary elements/insulators
Keeps two closely located genes separately |
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Term
| General Transcription Factors in Euk |
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Definition
| TFIID, TFIIB, TFIIF, TFIIE, TFIIH, TFIID |
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Term
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Definition
o Very complex
o Recruited along with TFIIA and TFIIB
o Contains many subunits
o Transcriptional coactivator
o Bridge between the enzymes and TF |
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Term
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Definition
o Put a large number (~100K) of cDNA sequences or synthetic DNA oligomers onto a glass slide (or other subtrate) in known locations on a grid.
o Target mRNA cDNA via reverse transcriptase
o Label an DNA sample and hybridize
o Measure amounts of RNA bound to each square in the grid
o Make comparisons
Cancerous vs. normal tissue
Treated vs. untreated
Time course
o Many applications in both basic and clinical research |
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Term
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Definition
• 10% of African Americans have polymorphic alleles of Glucose-6-phosphate dehydrogenase that lead to haemolytic anemia when they are given the anti-malarial drug primaquine.
• AZT and cytochrome P450
o AZT needs to be modified by human enzyme before it can block HIV replication.
o One enzyme that metabolizes a large number of this pre-drug is cytochrome P450.
o 10% of the Caucasian population is homozygous for alleles of the Cytochrome P450 gene CYP2D6 that do not metabolize the hypertension drug debrisoquine, which can lead to dangerous vascular hypotension.
• Succinylcholine Toxicity
o 0.04% of individuals are homozygous for alleles of psedocholineseterase that are unable to inactivate the muscle relaxant drug succinylcholine, leading to respiratory paralysis.
• Isoniazid Metabolism
o There are many polymorphic alleles of the N-acetlytransferase (NAT2) gene with reduced (or accelerated) ability to inactivate the drug isoniazid.
o Some individuals developed peripheral neuropathy in reaction to this drug
o Some alleles of the NAT2 gene are also associated with susceptibility to various forms of cancer
• ACE |
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Term
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Definition
o Exons from different pre-mRNA
o RARE in mammals
o No lariat structure, forms a Y instead |
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Term
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Definition
o An exon shuffles to a new gene
Not mRNA
o Can be found in an unrelated gene
o Gene disruption most likely in intron
o Splicing mechanism leads to a new gene |
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