Transcription 3 Flashcards
4 Genes in Lac Operon?
LacI (Allosteric Inhibitor)
LacZ (Beta-galactosidase)
LacY (Permease)
LacA (transacetylase)
How does the lac inhibitor inhibit transcription?
Occludes RNA Pol from binding promoter. inhibitor binds as a tetramer to three operator sequences and bends the DNA.
Function: Adenylate Cyclase
convert ATP to cAMP, inhibited by glucose
How is adenylate cyclase regulated?
Activity is turned off when glucose binds.
Function: CAP
Catabolite Gene Activator Protein. Binds cAMP and binds to DNA. Stabilizes RNA Pol promoter binding by interacting with alpha-CTD
How does the cell sense levels of Nitrogen?
By modulating NtrB activity through the transition of α-ketoglutarate to glutamate.
Function: NtrB
Senses levels of nitrogen. Phosphorylates NtrC when nitrogen levels are high.
Function: Sigma 54
Binds to core polymerase and forms holoenzyme at specific promoters, but does not initiate open complex conformation until NtrC ATPase activity is activated.
Function: NtrC
Enhancer Binding Protein (EBP), Has ATPase activity that changes conformation of sigma 54
Mechanism: Nitrogen Sensing Gene Regulation Pathway
1) NtrB recognizes high nitrogen levels and phosphorylates NtrC
2) NtrC-P bound to UAS is able to bind to sigma 54 by DNA bending facilitated by IHF
3) NtrC-ATPase activity causes conformational change in sigma 54
4) Conformational change in sigma 54 causes open complex formation
What part of transcription initiation does the Lac inhibitor and CAP effect?
RNA Polymerase Promoter Binding
What part of transcription initiation does Sigma 54 and NtrC effect?
Open complex formation
What GTF regulates lymphocyte transcription?
TFIIH
Technique: ssDNA-seq
Use KmNO4, modifies thymidine in ssDNA to prevent re-annealing
use nuclease that cleaves ssDNA
biotinylate, sonicate, and sequence, can see where txn bubble is
How does TFIIH cause gene activation in activated lymphocytes?
In resting cells, RNA Pol is bound to the promoter, but in a closed complex. When lymphocytes are activated, , TFIIH expression goes up. TFIIH can melt DNA when TFIIE binds it, get transcription
Function: TRF2
Alternative TBP that binds to TCT promoter
Function: TCT
Alternative to TATA box and is commonly found in ribosomal protein gene promoters. Bound by TRF2
Function: NF-KB (kappa beta)
Dimer of REL-family proteins, p50 and p65. Acts as a TF in the interferon induction pathway.
Function: I-KB (kappa beta)
Sequesters NF-KB in the cytoplasm to inactivate it.
Mechanism: NF-KB Regulation and Interferon Signaling
1) Signaling molecule binds receptor and activates downstream pathway
2) IKK phosphorylates I-KB
3) Phosphorylated I-KB is unbiquitinated and degraded
4) Degradation of I-KB releases NLS on NF-KB
5) NF-KB goes to nucleus to and turns on genes.
What happens to nucleosomes when a gene is being induced?
The nucleosome shifts to allow RNAP binding to the promoter. Coregulators bind enhancer sequence and recruit HATs and METs. ATP-dependent chromatin remodeling complexes move histones.
What are the two developmental activators in fruit flies?
Bicoid and Hunchback
What are the two developmental repressors in fruit flies?
Kruppel and Giant
How is even-skipped TF regulated in developing embryos?
Transcription of eve is reglated by multiple enhancers. Each enhancer has multiple binding sites for activators and repressors that are overlapping. Since the activators and repressors are expressed spatially within the embryo, different combinations of factors binding will allow certain enhancers to be active.
What are the 4 master regulatory factors that activate induced pluripotent stem cells?
Oct4, Sox2, Klf4, c-Myc
How was Oct4 first characterized?
Cells that underwent the first round of differentiation lost this factor.
How does Oct4 bind to DNA?
has two connected homeodomains, with each domain recognizing 4 bps in the major groove
Function: Sox2
binds with Oct4 (pioneer factor) and helps turn on transcription of NANOG
Function: NANOG
With OCT4 and SOX2, activates transcription of ES cell genes
How does Oct4, Sox2, and NANOG main pluripotency in stem cells?
The proteins act as positive transcription factors for their own genes, leading to an increase in their own expression.
What are super enhancers?
Enhancers that have a higher number of enhancer bound proteins, such as Med and Oct4, Sox2, NANOG.
Define expression of genes for differentiation.
Function: CTCF
Insulator protein. 11 Zn finger protein that binds directionally to DNA, found at base of loops that insulate enhancer function
Mechanism: Loop Extrusion Model for Insulation
Cohesin binds DNA and motor will push DNA through cohesin. Once two CTCF motifs with CTCF come into contact, extrusion will stop and enhancer sequence will be insulated.
