Lecture 4 - Regulation of gene expression 1

Question 1

What are cis-acting sequences?

Answer 1

• sequences of DNA that do not code for a transfusable product
• DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression

Question 2

What are trans-acting σ factors?

Answer 2

-factors, usually considered to be proteins, that bind to the cis-acting sequences to control gene expression

Question 3

What is a repressor?

Answer 3

• protein that binds to a specific sequence of DNA to block transcription
• acts to negatively regulate (with the operator)

Question 4

What is the operator?

Answer 4

-sequence of DNA where repressor binds

Question 5

What are transcription factors?

Answer 5

• proteins that interact with RNA pol and promote transcription
• acts to positively regulate

Question 6

What occurs in negative regulation of transcription?

Answer 6

A transacting repressor binds to the cis acting operator to turn off transcription

Question 7

What occurs in positive regulation of transcription?

Answer 7

-trans acting factors must bind to cis acting sites in order for RNA pol to initiate transcription at the promoter

Question 8

What is the concept of an operon?

Answer 8

• a cluster of 2 or more genes which are transcribed into a single mRNA which is polycistronic
• controlled by a single transcription unit
• normally encode proteins induced in the same function, which each gene normally located adjacent to one another e.g. LacZ, YA in the lac operon

Question 9

What are the features of the lac operon?

Answer 9

• lacI (repressor)
• LacZ (β-galactosidase (cleaves lactose to galactose and glucose)
• LacY (permease (responsible for intake of lactose into the cell)
• LacA (transacetylase)
• expression of these genes is controlled by a repressor (coded by LacI gene)
• repressor binds to the LacZYA operator (thich operlaps the promoter) when lactose is NOT present
• prevents expression of the enzymes as they are not needed and so their expression would waste energy

Question 10

What does the induction of the lac operon require?

Answer 10

• the synthesus of lacZYA enzymes in response to the appearance of lactose in the cell
• expressed at a basal level when lactose (a β-galactoside) is not present
• addition of lactose induces transcription of all three genes in the operon

Question 11

How come when lactose is removed from the external environment, expression of the lac enzymes quickly drop?

Answer 11

the lacmRNA is extremely unstable and quickly degraded

protein mpre stable than mRNA and so protein elvels remain higher for longer

Question 12

By what process is the lac repressor controlled by a small molecule inducer?

Answer 12

-repressor has two binding sites: one for the operator of DNA and one for the inducer
-Binding of the inducer changes the conformation of the repressor
-so that it can no longer bind to the operator on DNA
THIS IS AN EXAMPLE OF ALLOSTERIC CONTROL
(the main inducer molecule used in the lab is IPTG (isopropyl thiogalactosidase))

Question 13

What occurs when there is a mutation in the operator of the lac operon?

Answer 13

• mutations in the operator cause constitutive expression of all three lac strucural genes
• repressor is still produced but can’t bind to the operator as it is mutated
• mutations are cis-acting and only affect these genes on the continuous stretch of DNA
• transcription occurs with or without lactose
• constitutive expression

Question 14

What types of mutations identified the repressor gene, and what are the features of a mutation in the lacI gene?

Answer 14

-trans-acting mutations
Mutations in the lacI gene:
-are trans-acting
-affect expression of all the lacZYA clusters in the bacterium
Mutations that eliminate lacI function:
-cause constituative expression
-are recessive (shown: have cell diploid for lac genes (one on plasmid as well as in nuclear DNA) if the gene is WT will not get transcription of LacZYA as LacI protein is a trans factor so can diffuse across to work where needed)

Question 15

What results from a mutation in the DNA binding site of the repressor?

Answer 15

constitutive expression

-defective repressor cannot bind to the operator

Question 16

What results from mutations in the inducer binding site of the repressor?

Answer 16

-prevents the repressor being inactivated, causing uninducability, never get transcription

Question 17

What results in mutations in the lac promoter?

Answer 17

• cis acting
• RNA polyerase can’t bind regardless of lactose presence
• causes uninducability

Question 18

What is the structure of the lacI repressor?

Answer 18

-homo-tetramer
-contains two DNA binding subunits which are composed of two monomers each
-subunits dimerise to form a tetramer that can contact two operator sequences
Each monomer consists of 4 distinct regions:
-N terminal DNA binding domains (contains 2 short α helical regions which bind to the major groove of DNA)
-Hinge helix
-core domain
The inducer binding site is located in a pocket between core 1 and 2

Question 19

How does the repressor bind DNA?

Answer 19

• DNA binding domain of each monomer within a dimer inserts into the major groove of DNA
• the hinge helix inserts into the minor groove of the operator DNA

Question 20

How is repressor DNA binding regulated in the expression of the lac operon?

Answer 20

• regulated by an alloseric change in conformation
• with no inducer, the active repressor has a conformation in which the 2 DNA-binding domains of a dimer can insert into the successive turns of the double helix
• when the inducer binds it disrupts the hinge and changes the conformation, meaning that the 2 DNA binding sites no longer in the right geometry to make simultaenous contacts

Question 21

What is the structure of the operator sequence in the lac operon?

Answer 21

• symmetrical
• consists of 2 inverted repeats (palindromic sequence, 26bp long)
• repressor binds to the operator dsDNA sequence
• each monomer of the repressor contacts one 1/2 site

Question 22

What is a palindromic sequence?

Answer 22

DNA sequence reads the same on each strand when read in the 5’ to 3’ direction

Question 23

What is required for full repression of the lac operon?

Answer 23

LacOperon actually has 3 operators, O3 (88bp), O1, O2 (410 bp)
For full repression the repressor must bind to:
-O1 (operator at the lacZ promoter)
-an additional operator downstream (O2) or upstream (O3)
-each dimer in a repressor tetramer binds one operator, so that the tetramer binds 2 operators simaeltaeously
-binding of two operators causes DNA looping to prevent transcription at the promotor

Question 24

How is the lacoperon itself regulated (regardless of the operator/repressor)?

Answer 24

CRP activator protein (cyclic AMP receptor protein)

• CRP dimer is active when cAMP binds to it
• CRP dimer binds to a target sequence adjacent to the promoter
• CRP introduces a 90* bend in the DNA at its binding site
• this bend allows RNA pol to bind and for transcription to occur