Transcription 3

Question 1

4 Genes in Lac Operon?

Answer 1

LacI (Allosteric Inhibitor)
LacZ (Beta-galactosidase)
LacY (Permease)
LacA (transacetylase)

Question 2

How does the lac inhibitor inhibit transcription?

Answer 2

Occludes RNA Pol from binding promoter. inhibitor binds as a tetramer to three operator sequences and bends the DNA.

Question 3

Function: Adenylate Cyclase

Answer 3

convert ATP to cAMP, inhibited by glucose

Question 4

How is adenylate cyclase regulated?

Answer 4

Activity is turned off when glucose binds.

Question 5

Function: CAP

Answer 5

Catabolite Gene Activator Protein. Binds cAMP and binds to DNA. Stabilizes RNA Pol promoter binding by interacting with alpha-CTD

Question 6

How does the cell sense levels of Nitrogen?

Answer 6

By modulating NtrB activity through the transition of α-ketoglutarate to glutamate.

Question 7

Function: NtrB

Answer 7

Senses levels of nitrogen. Phosphorylates NtrC when nitrogen levels are high.

Question 8

Function: Sigma 54

Answer 8

Binds to core polymerase and forms holoenzyme at specific promoters, but does not initiate open complex conformation until NtrC ATPase activity is activated.

Question 9

Function: NtrC

Answer 9

Enhancer Binding Protein (EBP), Has ATPase activity that changes conformation of sigma 54

Question 10

Mechanism: Nitrogen Sensing Gene Regulation Pathway

Answer 10

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

Question 11

What part of transcription initiation does the Lac inhibitor and CAP effect?

Answer 11

RNA Polymerase Promoter Binding

Question 12

What part of transcription initiation does Sigma 54 and NtrC effect?

Answer 12

Open complex formation

Question 13

What GTF regulates lymphocyte transcription?

Answer 13

TFIIH

Question 14

Technique: ssDNA-seq

Answer 14

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

Question 15

How does TFIIH cause gene activation in activated lymphocytes?

Answer 15

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

Question 16

Function: TRF2

Answer 16

Alternative TBP that binds to TCT promoter

Question 17

Function: TCT

Answer 17

Alternative to TATA box and is commonly found in ribosomal protein gene promoters. Bound by TRF2

Question 18

Function: NF-KB (kappa beta)

Answer 18

Dimer of REL-family proteins, p50 and p65. Acts as a TF in the interferon induction pathway.

Question 19

Function: I-KB (kappa beta)

Answer 19

Sequesters NF-KB in the cytoplasm to inactivate it.

Question 20

Mechanism: NF-KB Regulation and Interferon Signaling

Answer 20

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.

Question 21

What happens to nucleosomes when a gene is being induced?

Answer 21

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.

Question 22

What are the two developmental activators in fruit flies?

Answer 22

Bicoid and Hunchback

Question 23

What are the two developmental repressors in fruit flies?

Answer 23

Kruppel and Giant

Question 24

How is even-skipped TF regulated in developing embryos?

Answer 24

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.

Question 25

What are the 4 master regulatory factors that activate induced pluripotent stem cells?

Answer 25

Oct4, Sox2, Klf4, c-Myc

Question 26

How was Oct4 first characterized?

Answer 26

Cells that underwent the first round of differentiation lost this factor.

Question 27

How does Oct4 bind to DNA?

Answer 27

has two connected homeodomains, with each domain recognizing 4 bps in the major groove

Question 28

Function: Sox2

Answer 28

binds with Oct4 (pioneer factor) and helps turn on transcription of NANOG

Question 29

Function: NANOG

Answer 29

With OCT4 and SOX2, activates transcription of ES cell genes

Question 30

How does Oct4, Sox2, and NANOG main pluripotency in stem cells?

Answer 30

The proteins act as positive transcription factors for their own genes, leading to an increase in their own expression.

Question 31

What are super enhancers?

Answer 31

Enhancers that have a higher number of enhancer bound proteins, such as Med and Oct4, Sox2, NANOG.

Define expression of genes for differentiation.

Question 32

Function: CTCF

Answer 32

Insulator protein. 11 Zn finger protein that binds directionally to DNA, found at base of loops that insulate enhancer function

Question 33

Mechanism: Loop Extrusion Model for Insulation

Answer 33

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.