Transcriptional Control of Gene Expression 1 (L10)

Question 1

CAP

Answer 1

catabolite activator proteins - bind recognition site and stabilize binding of RNA pol to promoter -> increases transcription

Question 2

what does CAP depend on?

Answer 2

needs cAMP to bind

Question 3

sigma factor

Answer 3

an IF - different sigma factors used for transcribing different genes

Question 4

two functions of enhancers

Answer 4

1. spatial-temporal regulation of gene expression

2. alteration of expression level

Question 5

how do enhancers work?

Answer 5

by stabilizing RNA pol II binding via mediator proteins

Question 6

is an enhancer’s location important for its function?

Answer 6

not often

Question 7

how do enhancers bind regulatory proteins?

Answer 7

multiple recognition sequences - i.e. TFs (or activators/repressors)

Question 8

what does the proximal/basal promoter always bind?

Answer 8

always bind same TFs

Question 9

what does promoter consist of?

Answer 9

enhancers + basal promoters (DNA)

Question 10

what do activators consist of?

Answer 10

protein

Question 11

difference b/w enhancers and proximal promoter

Answer 11

enhancers can be all over; prox promoter is right before the gene

Question 12

UAS

Answer 12

upstream activating sequence - in yeast, equivalent of enhancer

Question 13

mammalian numbers of promoters and enhancers

Answer 13

only one promoter but multiple enhancers

Question 14

spatial-temporal regulation

Answer 14

control where genes are expressed and when expressed during life cycle

Question 15

what are enhancers?

Answer 15

regulatory sequences (or promoter elements) that are bound by TFs - aid in signal transduction

Question 16

TFBS

Answer 16

TF binding site - in one enhancer, can have many TFs binding at same TFBS

Question 17

TATA box

Answer 17

consensus sequence - same sequence found more often than just randomly would occur - important regulatory sequence for RNA pol

Question 18

CpG island

Answer 18

20-50 nts within 100 bps upstream

Question 19

functions of RNA pol I

Answer 19

• ribosome components
• protein synthesis
• binds basal promoter elements

Question 20

functions of RNA pol II

Answer 20

• transcribes mRNA, miRNA, siRNA
• encodes protein
• RNA splicing
• post-translational gene control

Question 21

functions of RNA pol III

Answer 21

• protein synthesis
• ribosome component, protein synthesis
• RNA splicing
• signal-recognition particle for insertion of polypeptides into the ER
• various unknown functions

Question 22

CTD

Answer 22

C-terminal domain on beta1 subunit of RNA pol II - consists of 52 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (phosphorylation)

Question 23

how can you differentiate when CTD is phosphorylated or not?

Answer 23

phosphorylated form = red -> more decondensed areas
dephosphorylated form = green
both = yellow

Question 24

polytene chromosome

Answer 24

giant chromosome used to study regulation of gene expression

Question 25

experimental definition of promoter elements - two ways

Answer 25

1. deletion analysis

2. linker scanning mutation/deletion

Question 26

deletion analysis

Answer 26

Chop promoter into smaller pieces or use probes to replicate smaller pieces.
1. use recombinant DNA techniques to create a 5’ deletion series.
2. Ligate into vector carrying reporter gene.
3. Transform into E. coli and isolate plasmid DNAs.
4. Transfect each type of plasmid (1-5) separately into cultured cells.
5. Prepare cell extract and assay activity or reporter enzyme.
Reporter gene expression levels tell you which areas are necessary for expression.

Question 27

what does deletion analysis tell you?

Answer 27

where important regions of promoter are - useful for analyzing basal promoter region

Question 28

difference b/w deletion analysis and linker scanning mutation/deletion analysis

Answer 28

deletion: chops from end
linker: chops in middle in overlapping way

Question 29

linker scanning mutation/deletion

Answer 29

same principles as deletion analysis, but chops in middle in overlapping way - allows for better determination of where the important elements are

Question 30

what happens if you delete the TATA box?

Answer 30

RNA pol can’t bind at all - no expression

Question 31

what happens if you delete other regulatory elements than the TATA box?

Answer 31

these just help stabilize binding of RNA pol, so if deleted, just get a decrease in expression

Question 32

EMSA

Answer 32

electrophoretic mobility shift assay/gel shift
1. mix DNA probe with protein fractions from chromatography
2. run on gel
If protein does NOT bind probe, runs faster.
If protein DOES bind probe, lower mobility - slower migration. Any lanes with slow migrating bands - know there is a protein in this fraction that can bind promoter

Question 33

what does yeast-1-hybrid test?

Answer 33

interaction b/w protein and DNA - if protein from cDNA library can bind to promoter element -> get expression of that construct

Question 34

how do you verify potential identification of DNA-binding proteins?

Answer 34

1. Purify the protein by sequence-specific DNA affinity chromatography.
2. Sequence protein and clone the gene.
3. Insert a plasmid containing the gene sequence for what you think is protein X and a plasmid containing a reporter gene and a binding site for protein X into a cell.
4. If the gene produces protein that binds to the X-binding site, then transcription of the reporter gene will occur (means your protein truly binds the promoter)

Question 35

difference b/w yeast-1-hybrid and yeast-2-hybrid

Answer 35

1: 1 construct, tests protein-DNA interaction
2: 2 constructs (bait/prey), tests protein-protein interaction

Question 36

describe expression when you delete parts of AD

Answer 36

less expression than normal

Question 37

describe expression when you delete all of AD

Answer 37

no expression

Question 38

how can you define DBD and AD?

Answer 38

deletion mutants - N and C terminal deletion mutants or internal deletion mutants

Question 39

functional domains are…

Answer 39

modular

Question 40

how can TF’s be regulated?

Answer 40

some have activation domains, some have repression domains