Term
Describe RNA transcription
1. Polymerase
2. Promoter |
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Definition
1. A, G, U, C, Inosine, Xanthosine
5->3 direction de novo (no primer)
2. gene sequence to which polymerase binds
- in Prokaryotes, TTGACA -35 and TATAAT -10 |
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Term
| Describe DNA footprinting |
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Definition
1. DNA fragment is labeled with radioisotope or dye
2. Labeled DNA is incubated with RNA poly and then digested
3. Regions where poly binds to DNA can be determined |
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Term
Describe the lac operon
Z
Y
A
o
i |
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Definition
*each gene codes for the following proteins
Z: beta galactosidase- cleaves lactose
Y: lactose permease- allows lactose to enter cell
A: transacetylase- inactive toxic thiogalactosides that come into cell with lactose
o: operator- next to TSS, controls transcription
i: far away from operon, encodes for a protein that binds to the operator |
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Term
| Describe the process of the lac operon |
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Definition
*negative control
1. repressor encoded by i gene binds to operator and prevents poly from binding
2. lactose levels increase making allolactose available to bind to repressor-> falls of operator
3. poly is free to bind and transcribe operon |
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Term
| Describe cis and trans acting factors |
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Definition
Cis: only affect expression of genes on same strand
Trans: can affect genes located on another chromosome |
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Term
| Describe the role of glucose in transcription in E. coli |
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Definition
*positive control
1. glucose normally inhibits expression of genes for other sugars
2. low glucose active adenyl cyclase
-converts ATP to cyclic AMP
3. cAMP then binds to CAP
-CAP binds with poly and increases expression |
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Term
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Definition
-regulatory sequences that are far away from gene
-orientation and distance do not matter in regards to TSS
-bind to TFs that regulate RNA poly
-DNA loop allows distant enhancers to bind to TFs
ex. Ig enhancer contains negative regulatory elements that inhibit transcription and also positive that activate trans in B cells |
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Term
| Describe promoter regulatory sequences |
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Definition
-controls gene expression to downstream genes
-can be determined by placing a marker right after the regulatory sequence
ex. firefly luciferase |
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Term
| Describe how TF binding sites are identified |
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Definition
1. DNA foot-printing and electrophoretic mobility shift assay
-radio labeled DNA fragments are incubated with protein and then run in a gel
-migration of DNA fragment through the gel is slowed by bound protein
-TF binding sites are usually short DNA sequences and degenerate
2. DNA affinity chromatography
-dsOligos with GC repeats are bounds to agarose beads in column
-cell extracts pass through column and TF binds to GC region and is retained in the column
-TF binds and is retained while everything else passes through |
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Term
Describe DNA binding domains
1. Zinc fingers
2. Helix loop helix |
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Definition
*normally dimers that look for palindromes
1. binds zinc ions and folds into loop fingers that bind DNA
-Zn connects alpha helix and beta sheets
-steroid hormone receptors contain Zn fingers and regulate trxn in response to hormone
-most common
2. acts as dimer from different members of each family |
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Term
Describe DNA binding domains
3. Helix turn helix
4. Leucine zipper |
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Definition
3. one helix contacts DNA while others lay over top to stabilize
ex. homeodomain proteins (Hox) which regulate gene expression during embryonic development
4. forms a dimer with combinations of different families |
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Term
| Describe homeodomain proteins |
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Definition
-regulate expression in embryonic development
-highest level of control
-first seen in flies where legs grew out of head (Antennapedia)
-another Hox protein is Pax6 which controls eye development
-turns on all genes downstream from it that code for eye development
*master control gene=light switch |
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Term
| Describe repression in eukaryotes |
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Definition
-can be regulated by repressors that bind to DNA and block trxn
-interfere with TFs or compete with activators that bind to regulatory sequences |
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Term
| Describe the role of multiple TFs |
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Definition
different combinations of TFs can produce different structures and determine cell differentiation
ex. Bicoid and hunchback TF go together to form new phenotype
-cells that share similar lineage will have similar TFs
ex. mesoderm, endoderm, ectoderm |
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Term
| Describe X chromosome inactivation |
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Definition
-one of the two Xs are turned off
-RNA regulatory gene Xist binds and coats X and inactivates chromosome
-leads to chromosome condensation and conversion to heterochromatin |
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Term
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Definition
-methyl groups are added to 5C of CpGs
-transcription is repressed |
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Term
| Describe the stability of RNA |
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Definition
-RNAs are degraded into cytoplasm
-intracellular levels are determined by a balance between synthesis and degradation
-rate of degradation can control gene expression
Bacteria: rapidly degraded (2-3 mins)
-allows them to respond rapidly
Eukaryotes: half life varies (30mins-20hrs)
-short lived mRNAs are for regulatory proteins
-structural proteins and metabolic enzymes have longer lives |
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Term
| Describe the degradation of RNA |
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Definition
1. initiated by shortening of polyA tails
2. rapidly degraded mRNAs contain AU rich sequences near 3' end which are binding sites for proteins that stabilize or target them for degradation
3. RNA binding proteins are regulated by growth factors and hormones
-also regulated by siRNAs and miRNAs |
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