Transcriptional Control of Gene Expression 1

Question 1

What are the components of the lac operon of E. coli?

Answer 1

The CAP site, the lac promoter, and the lac operator

Question 2

Function of CAP site

Answer 2

Binds catabolite activator protein (Cyclic AMP binds to CAP, which binds to the CAP site)

Question 3

Function of lac promoter

Answer 3

binds the alpha70-RNA polymerase complex

Question 4

Function of lac operator

Answer 4

binds lac repressor

Question 5

In the absence of lactose, very little lac mRNA is produced because _____.

Answer 5

the lac repressor binds to the operator, inhibiting transcription initiation by
alpha70-RNA polymerase

Question 6

Maximal transcription of the lac operon occurs in the presence of ____.

Answer 6

lactose and absence of glucose

(In this situation, cAMP increases in response to the low glucose concentration and forms the CAP-cAMP complex, which binds to the CAP site, where it interacts with RNA polymerase to stimulate the rate of transcription initiation. )

Question 7

In the presence of glucose and lactose, lac

repressor binds _____.

Answer 7

lactose and dissociates from the operator, allowing alpha70-RNA polymerase to initiate transcription at a low rate

Question 8

What is the function of the sigmoid factor?

Answer 8

It brings RNA polymerase to the promoter.

Different sigma factors are used for transcribing different genes.

Question 9

TATA box

Answer 9

core (basal) promoter and enhancers

Question 10

Regulatory elements in mammalian and yeast cells

Answer 10

TATA box, Mammalian CpG-Island promoter gene, Upstream activating sequence (in yeast)

(Yeast is simple: TATA box and UAS)

Question 11

Mammalian genes with a TATA-box promoter are regulated by _____.

Answer 11

promoter-proximal elements and enhancers

Question 12

Mammalian CpG-Island promoters

Answer 12

Transcription initiates at several sites in both the sense and antisense directions from the ends of the CpG-rich region. Transcripts in the sense direction are elongated and processed into mRNAs by RNA splicing. They express mRNAs with alternative 5’ exons determined by the transcription start site. CpG-island promoters contain promoter-proximal control elements.

Question 13

TFIIB

Answer 13

Recognizes (G/C)(G/C)(G/A)CGCC element upstream of TATA box

Question 14

TATA box sequence

Answer 14

TATA(T/A)A(A/T)(A/G)

Question 15

Initiator sequence in mammals

Answer 15

YYAN(T/A)YY

Question 16

Length of CPG islands

Answer 16

on average 1000 bp long

Question 17

The core basal promoter is bound by ___, which anchor ___ on the promoter to begin transcription at the ___.

Answer 17

basal transcription factors,
RNA polymerase,
transcription start site

Question 18

RNA polymerase I transcribes ___.

Answer 18

Pre r-RNA

He said this wasn’t important to know but I saw past exam questions on it I thought.

Question 19

RNA polymerase II transcribes ____.

Answer 19

mRNA, snRNAs, siRNAs, and miRNAs

Question 20

RNA polymerase III transcribes ____.

Answer 20

tRNAs

Also 5S rRNA, snRNA U6, 7S RNA. Not important

Question 21

All three yeast polymerases have five core subunits homologous to the ___ of E. coli RNA polymerase.

Answer 21

beta, beta’(prime), two alpha, and omega subunits

Question 22

The largest subunit (RPBl) of RNA polymerase II also contains an essential ____.

Answer 22

C-terminal domain (CTD)

Question 23

Vertebrates have ___ repeats of Tyr‐Ser‐Pro‐Thr‐Ser‐Pro‐Ser .

Answer 23

52

Question 24

Transcription factors and what they bind to.

Answer 24

TBP binds to TATA box. Bends DNA.
TFIIB binds to TBP
TFIIF/Pol II complex binds TFIIB
TFIIE binds to TFIIF/Pol II complex
TFIIH binds to TFIIE and TFIIF
General factors except TBP released

Question 25

What does TBP do?

Answer 25

Binds to the TATA box and bends the DNA

(TBP is the first protein to bind to a TATA box promoter. All eukaryotic TBPs analyzed to date have very similar C-terminal domains of 180 residues. This domain of TBP folds into a saddle-shaped structure; the two halves of the molecule exhibit an overall dyad symmetry but are not identical. TBP interacts with the minor groove in DNA, bending the helix considerably (see Figure 4-5). The DNA-binding surface of TBP is conserved in all eukaryotes, explaining the high conservation of the TATA box promoter element )

Question 26

Transcription pause after 20‐50 nucleotides caused by ____ and is relieved by ___.

Answer 26

binding with NELF (negative elongation factor)
(Or DSIF (DRB sensitivity‐inducing factor)/ Spt4/5)

Relieved by Cdk9/Cyclin T
(P‐TEFb/positive transcription elongation factor b)
(Phosphorylate Ser 2 of the CTD
Relationship with AIDS; Tat regulation of Cdk9/Cyclin T activity)

Question 27

Activators are ____ that bind to ___.

Answer 27

Transcription factors,

enhancers

Question 28

Mediators are ___.

Answer 28

protein complexes that act as a molecular bridge between TFs and basal TFs

(Different TFs bind different subunits)

Question 29

The integration of signals from different ____ determines the tissue specific output as well as the output level.

Answer 29

Transcription factors on the promoter/enhancers

Question 30

What assay is used to study function of regulatory elements?

Answer 30

Reporter assay

Shorter and shorter DNA sequences used, see if protein is made or not in bacteria after plasmid is taken up

Question 31

Linker Scanning/Deletion

Answer 31

Different segments of DNA taken out to find promoters, enhancers, inhibitors (what’s necessary, what’s not, where they are)

Question 32

Comparative genomics

Answer 32

Can use beta-galactosidase (reporter gene) in area of gene of interest to see where it’s expressed

Question 33

EMSA or gel shift

Answer 33

In this example, protein fractions separated by column chromatography were assayed for their ability to bind to a radiolabeled DNA-fragment probe containing a known regulatory element. After an aliquot of the protein sample was loaded onto the column (ON) and successive column fractions (numbers) were incubated with the labeled probe, the samples were electrophoresed under conditions that do not disrupt protein-DNA interactions. The free probe not bound to protein migrated to the bottom of the gel. A protein in the preparation applied to the column and in fractions 7 and 8 bound to the probe, forming a DNA-protein complex that migrated more slowly than the free probe. These fractions therefore likely contain the regulatory protein being sought.

Question 34

Yeast-1 assay

Answer 34

One protein, see if transcription occurs

Question 35

Know about GAL4

Answer 35

Found UAS, stuff like that. I’m running out of time to look at slides so I’m done with this.