Regulation of transcription - eukaryotes

Question 1

What can regulate gene expression/transcription initiation?

Answer 1

1. Sigma factor - diff ones dictate which promoters are on/off.
2. Strength of promoter.
3. Regulatory proteins - usually DNA binding proteins.
   a. Repressors - inhibit transcription when bound to DNA.
   b. Activators - elevate “”
   c. Dual function regulators - do either depends on conditions

Question 2

What regulates the binding of activators/repressors to DNA?

Answer 2

Ligands

Question 3

What do inducers do?

Answer 3

They are ligands that bind to and inactivate repressors.

Question 4

What do corepressors do?

Answer 4

Ligands that bind to and activate repressors.

Question 5

In an activator where the ligand is an inducer joins with the activator when will there be transcription?

Answer 5

When the activator is bound to the DNA as the inducer induces the transcription.

Question 6

When the ligand acts as a repressor with an activator when will there be transcription?

Answer 6

When only the activator is bound to DNA as the repressor ligand will remove it from the DNA so transcription can’t occur.

Question 7

When the ligand acts as a corepressor with a repressor when will there be transcription?

Answer 7

When the repressor is not joined with the co-repressor and not on the DNA. The co-repressor helps it repress.

Question 8

When the ligand is an inducer with a repressor when will there be transcription?

Answer 8

When the inducer is bound to the repressor not on the DNA. The inducer essentially represses the repressor stopping it binding to DNA so transcription can occur.

Question 9

What happens in bacteria when glucose is not present as a carbon source?

Answer 9

Lactose is used as an indirect carbon source.

Question 10

What happens to the levels of enzymes when lactose is used as a carbon source?

Answer 10

Increase dramatically when lactose is present as enzymes are inducible (produced in response to the presence of an appropriate inducer) and lactose=inducer.

Question 11

What is polycistronic mRNA?

Answer 11

An mRNA corresponding to multiple genes whose expression is also controlled by a single promoter and a single terminator. Polycistronic mRNAs are also called operons. All eukaryotic mRNAs are monocistronic.

Question 12

What does the Beta-galactosidase enzyme do?

Answer 12

AKA lacZ gene
Enzyme
Converts lactose to galactose and glucose

Question 13

What does the lacY gene do?

Answer 13

Permease - entry of lactose into cell.

Question 14

What does lacA do?

Answer 14

Transacetylase - Any enzyme that catalyses the transfer of an acetyl group from one molecule to another. It is produced by the ‘a’ structural gene of the lac operon.

Question 15

How is the lac operon regulated?

Answer 15

The lacI repressor - repressor tetramer binds to the operator and prevents the RNA polymerase from binding to the promoter.
No transcription.

Then lactose (inducer) binds to the tetramer causing conformational change (4 inducers needed).
Repressor leaves so transcription can occur. RNA pol can bind.
IPTG is also used as a (non-physiological) inducer.

Question 16

Is the lac operator pallindromic?

Answer 16

Yes - sequence mutants are often consitutive.

Question 17

Do DNA binding proteins often act as monomers?

Answer 17

No they tend to act as dimers - the palindromic sequence helps this happen.

Question 18

Is LacI a corepressor?

Answer 18

No it is an inducer

Question 19

What is the structure of the LacI repressor?

Answer 19

Head - DNA binding helix-turn-helix.
Hinge
Core domain 1
IPTG binding site
Core domain 2
Oligomerisation domain - alpha helical stretch/domain that promotes dimer formation.

Question 20

What happens to lacI repressor in the presence of lactose.

Answer 20

Conformational change is induced so DNA binding is abolished (transcription occurs).

Question 21

How does the LacI repressor inhibit transcription?

Answer 21

DNA looping -tetramer - 2 bind to diff lac operators

Question 22

What prevents lactose being broken into glucose and galactose?

Answer 22

Catabolite Activating Protein (CAP)

Question 23

Is the lac promoter strong?

Answer 23

No it is pretty weak.

Question 24

What does the CAP do to RNA pol?

Answer 24

C terminal domain
CAP pulls RNA pol towards it.

Question 25

What happens when CAP is inactive?

Answer 25

No ligand binding so RNA pol is not able to start transcription. This is in the presence of glucose - no cAMP (inducer)

Question 26

What does cAMP do to inactive CAP?

Answer 26

It makes it active so it can bind to CAP site and RNA pol can then bind to DNA.

Question 27

What are some facts about CAP?

Answer 27

1. activates transcription at more 100 promoters.
2. MM = 45 kDa and binds DNA as a dimer.
3. CAP-depenedent promoters only have 3 componets requires: CAP, RNA pol and DNA. Most bacterial/eukaryotic promoters are more complicated.

Question 28

What does CAP need for it to work?

Answer 28

Needs to be on the same side as the RNA pol.
No CAP-dependent promoter has a good -35 site.

Question 29

WHat does CAP do for gal promoter?

Answer 29

CAP increases the transition from the closed–> open promoter.

Question 30

What does the CAP do for the lac promoter?

Answer 30

Increases the formation of the closed promoter complex.

Question 31

What does CAP recruit and how?

Answer 31

Recurits: RNA pol
By making contact w/ alpha-subunit (a-CTD)

Question 32

What are the diff classes of CAP-dependent promoter?

Answer 32

(class) I
II
III

Question 33

What happens in CAP class I mechanism?

Answer 33

Interactions btwn CAP and a-CTD increase binding constant for formation of the closed promoter complex. Increases initiation by facilitating recruitment of holoenzyme to the promoter.
Note: a-CTD binds to DNA immediately downstream of CAP in both cases.

Question 34

What happens in CAP class II?

Answer 34

Only requires CAP for activation and have a single CAP site that overlaps the promoter, replacing the -35 site.
Upstream CAP makes contact w/ a-CTD w/ AR1.
The downstream CAP makes contact w/ N-terminal domain of a through AR2.
a-CTD makes contact w/ DNA upstream of CAP. CAP is located in its preferred position.
Interaction facilitates an isomerisation that results in an increase in the rate of open complex formation (promoter melting).

Question 35

What happens in CAP class III?

Answer 35

Requires 2+ CAP dimers/ 1+ CAP molecules w/ 1+ regulon-specific activators

Question 36

What does MerR do to transcription?

Answer 36

Activates transcription w/out contacting RNAP.
Controls merT gene (encodes enzyme makes cell resistant to toxic effects of mercury)
Binds betwn -10 and -35 region of merT promoter. In presence of mercury, MerR activates merT.
Confirmation change -10 changes position.

Question 37

What does trp operon encode for and when?

Answer 37

5 contiguous structural genes required for tryptophan synthesis.
Only expressed when tryptophan is limiting.

Question 38

What are the levels involved in trp operon?

Answer 38

2 lvlvs of regulation involved:
1- transcription repression by the trp repressor (regulation of initiation). tryptophan = corepressor (binds and stops mRNA being transcribed)
2- attenuation (regulation after the initiation). No trp = long trp mRNA transcribed. Trp= present = short mRNA produced.