Study Guide/Outline—Bacterial Gene Regulation Bacterial Gene Regulation What is an operon? How is...

Study Guide/Outline—Bacterial Gene Regulation

Bacterial Gene Regulation• What is an operon? How is it different from a eukaryotic gene?• In the lac operon, what cellular or environmental conditions must exist in

order for the (WT) lac operon to express its genes? How do these environmental conditions positively or negatively regulate the operon?

• What are the different parts, and their functions, of the operon? • How do mutations in “upstream” parts of the operon (promoter, operator,

coding genes) affect the “downstream” areas of the operon? How do missense and nonsense mutations have different results?

• The lacI gene is not part of the Lac Operon. How is the lac I gene involved with the Lac operon?

• What kinds of mutations are cis-dominant? Trans-dominant? Constitutive ON? Constitutive-OFF?

• How can a bacteria be a partial diploid? How does being diploid for the LacI gene create complexities in the regulation of the Lac Operon?

Brooker Fig 16.3b

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HH

H

H

OH

OHHO

HO

CH2OHO

HOH

Galactose

H

H

H

OH

OHH

CH2OHO

HOH

HH

H

H

OH

HO

CH2OHO

HOHH

H

H

O

OHH

CH2OHO

OHOH

HH

H

H

OH

HO

HO

CH2OHO

HOHH

H+

H+

H

H

OH

OHH

CH2

OO

HOH

Glucose

+

galactosidase

galactosidase

βgalactosidaseside reaction

Allolactose

Lactose

Cytoplasm

LactoseLactose permease

Functions of lactose permease and -galactosidase

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Brooker Fig 16.8

PromoterCAP site Operator

(b) No lactose or glucose (high cAMP)

cAMP

Transcription is very lowdue to the binding of therepressor.

Repressor

CAP

cAMP

CAP

PromoterCAP site Operator

Repressor(inactive)

High rate of transcription

Allolactose

Binding of RNA polymeraseto promoter is enhancedby CAP binding.

(a) Lactose, no glucose (high cAMP)

Positive control—Catabolite Activator Protein (CAP) turns on Lac Operon

High rate of transcription

But negative control Must be removed before positive control will result in transcription

Brooker, Fig 16.8

Allolactose

PromoterCAP site Operator

Repressor(inactive) (Inactive)

(Inactive)

(c) Lactose and glucose (low cAMP)

PromoterCAP site Operator

(d) Glucose, no lactose (low cAMP)

Transcription rate is lowdue to the lack of CAPbinding.

CAPTranscription is very low dueto the lack of CAP binding andthe binding of the repressor.

CAP

In absence of cAMP, transcription is very low (or hardly at all)

lac repressor bindsto the operator andinhibits transcription.

lacregulatorygene

lac operon

mRNA

lacI lacP lacO lacZ lacY lacA

lac repressor(active)

(a) No lactose in the environment

Figure 16.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Constitutive expression of

lacI

Promoter Operator

RNA pol cannot access the promoter when repressor bound to operator

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Figure 16.4

RNA polymerase

mRNA

lacI lacP lacO lacZ lacY lacA

Allolactose

Transcription

(b) Lactose present

galactosidase Lactosepermease

Galactosidetransacetylase

The binding of allolactose causes aconformational change that preventsthe lac repressor from binding to theoperator site.

Conformationalchange

PolycistronicmRNA

Lactose causes repressor to fall off Operator Site

Figure 16.5a

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Lac repressor

Lactose permease

galactosidase Lactose

Lac repressor

Transacetylase

4. Most proteins involvedwith lactose utilizationare degraded.

1. When lactose becomesavailable, a small amount of itis taken up and converted toallolactose by β-galactosidase.The allolactose binds to therepressor, causing it to fall offthe operator site.

2. lac operon proteinsare synthesized. Thispromotes the efficientmetabolism of lactose.

3. The lactose is depleted.Allolactose levels decrease.Allolactose is released fromthe repressor, allowing it tobind to the operator site.

Lac repressor

lac

operon

lac

operon

lac

operon

lac

operon

Induction of Lac Operon

Animation Lac Operon

http://vcell.ndsu.nodak.edu/animations/

http://vcell.ndsu.nodak.edu/animations/

Brooker Figure 16.7

4. Incubate the cells long enough to allow lac operon induction.

5. Burst the cells with a sonicator. Thisallows β-galactosidase to escape fromthe cells.

– LactoseF’

F

In mero-zygote strain, the lac I+ gene on the F´ factormakes enough repressor to bind to both operator sites (restoring WT phenotype on main chromosome).

Lactose is taken up, is converted toallolactose, and removes the repressor.

1 2 3 4

Lactose

3.

+ Lactose

4.Z+

I–

P O Y+

A+ Z+

PO Y+

I+

Experimental level Conceptual level

1. Grow mutant strain and merozygote strain separately.

2.Divide each strain into two tubes.

3. In one of the two tubes, add lactose.

Mutantstrain

Merozygotestrain Merozygote

– Lactose

Mutant

F′

Operon is constitutive-on in Mutant strain becauseno repressor is made.

1.

+ Lactose

2.

Z+

I–

P O Y+

A+

Z+

I–

P O Y+

A+ Z+P O

Y+

A+I+

Z+

I–

PO Y +

A+

Z +

I–P O Y +

A +

A+

Z+

I–

P O Y+

A+ Z+P O Y+

I+ A +

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6. Add β-o-nitrophenylgalactoside (β-ONPG). This is a colorlesscompound. β-galactosidase willcleave the compound to producegalactose and o-nitrophenol (O-NP).O-NP has a yellow color. The deeperthe yellow color, the moreβ-galactosidase was produced.

7. Incubate the sonicated cells toallow β-galactosidase time tocleave β-ONPG.

8. Measure the yellow color producedwith a spectrophotometer. (Seethe Appendix for a descriptionof spectrophotometry.)

o-nitrophenyl-galactoside

O-NP

-galactosidase

Broken cell

1.

1 2 3 4

NO2NO2

+

2.

NO2NO2

+

3.

NO2

4.

NO2

+

ONPG Galactose

NO2

Brooker Figure 16.7, cont

Table 16.1 16 – 34

Question

Will a loss-of-function mutation in Plac (promoter sequence) be cis-dominant or trans-dominant?

Lactose status(assume absence of Glucose)

Genotype Promoter Seq

Repressor Operator Seq Lac Z Lac Y Lac A

Type of mutation (e.g. cis-dominant, consititutive ON)

Absent WT + Active BoundNo

ExpressionNo

ExpressionNo

Expressionnone

Present WT + Inactivated Open WT B-GalWT

PermeaseWT

Transacet.none

Present Lac Ymiss

Present Lac ZNons

Present P Lac(-) 

Absent Lac Oc

Present Lac Oc

Lac Z

Lactose status

(assume absence of Glucose)

GenotypePromoter

Seq Repressor Operator

Seq Lac Z LacY Lac A

Type of mutation (e.g. cis-

dominant)

Absent Lac I (-)              

AbsentF’-Lac I (+)

Lac I (-)             

AbsentF’-LacOc

Lac O+             

PresentF’-LacOc

Lac O+             

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