Regulation of Gene Expressionin Prokaryotes I

Question 1

so far we know…

Answer 1

• DNA is arranged into genes.
• Genes provide for the storage of information.
• This information is expressed through the processes of:
  Transcription
  Translation

Question 2

How is the expression of genes regulated such that we obtain a coordinated expression of the genetic material at the right time (and place) to obtain the desired effect?

Answer 2

• Very NB question
• Remember, not all genes are expressed at all times

Question 3

Efficient expression of genetic information

Answer 3

is dependant on control mechanisms that promote or suppress gene activities.

Question 4

How is the promotion/ suppression of gene expression achieved

Answer 4

In transcription:
The expression of genes relies on the presence of a cis-element termed a promoter and which is usually found upstream of the start codon of the gene.

Question 5

a promoter

Answer 5

Determines when and what quantities a gene will be transcribed

Question 6

Many of the early studies of gene expression where performed on bacteria and yeast
Why?

Answer 6

• They are easy to culture and have short generation times
• they can be easily mutated and pure mutated cultures can be
  obtained for separate studies

Question 7

Bacteria are ideal models for studies involving

Answer 7

• the induction of gene expression in response to changes in
  environmental conditions
• Bacteria regulate their gene expression in response to
  environmental changes as well as a variety of non-environmentally
  regulated cellular activities (such as cell division)

Question 8

Adaptive hypothesis

Answer 8

Refers to the ability of organisms to adapt to their environment
E.G:
lactose in growth medium induces expression of enzymes specific for lactose metabolism

Question 9

Various forms of gene expression

Answer 9

Constitutive, inducible, repressible systems

Which are under negative or positive control

Question 10

Constitutive genes

Answer 10

• permanently expressed, regardless of the environmental conditions
• Call these housekeeping genes - expressed all the time- keep cell
  alive

Question 11

Inducible genes

Answer 11

• expressed in response to a particular condition
  E.g: An inducer, such as lactose.

Question 12

repressible systems

Answer 12

• genes may be repressed due to the presence of a particular
  molecule
• These molecules are often end products of a specific biosynthetic
  pathway

Question 13

positive control

Answer 13

• needs a substrate to directly stimulate transcription

Question 14

Negative control

Answer 14

• occurs when a molecule turns off for transcription

Question 15

Glucose

Answer 15

is the primary molecule used as a source of energy in all cellular metabolism

Question 16

E. coli has the ability to grow on ______ as an alternative source of ______

Answer 16

lactose
carbon

Question 17

Lactose is a disaccharide of

Answer 17

Galactose and glucose

Question 18

Which enzyme digests lactose to produce galactose and glucose?

Answer 18

Beta-galactosidase

Question 19

In the absence of lactose

Answer 19

• There are only a few copies of the Beta-galactosidase enzyme
  present in the cell

Question 20

In the presence of lactose

Answer 20

• Gene expression is induced and the number of enzymes available
  rapidly increases to several thousand enzymes per cell
• The enzymes = inducable
• lactose = inducer molecule

Question 21

Lactose - the inducer

Answer 21

• technically, the inducer is allolactose - an isomer of lactose
• When lactose first enters the cell, a very small portion of it is
  converted to allolactose by the Beta-galactosidase enzyme

Question 22

operon

Answer 22

Prokaryotic genes with related functions are organised in groups and are expressed in a coordinated fashion

Question 23

Watch slide 11 of prokGenRegnarrated1

Question 24

The lac operon

Answer 24

• lac operon consists of 3 structural genes and
• 2 regulatory regions upstream (cis & trans action) - it is found 5’ to
  the operon
• The discovery of a regulatory gene and regulatory site which are
  not part of the gene cluster - NB in understanding how the operon
  was controlled
• neither of these regions encode enzymes needed for lactose
  metabolism = function of the 3 structural genes

Structural genes (related functions):
lacZ – b-galactosidase
lacY – b-galactoside permease
lacA – transacetylase

However, together, this entire gene cluster operates to provide a rapid response to the lactose status of the environment

Question 25

Structural genes (related functions):

Answer 25

Genes and what they encode:
lacZ – b-galactosidase
> responsible for cleaving lactose into glucose and galactose

lacY – b-galactoside permease
> facilitates the entry of lactose into the cell

lacA – transacetylase
> May be involved in the removal of toxic products of lactose
metabolism (unclear)

Question 26

Functions of these structural genes were identified through the study of lac minus mutants - first isolated and studied by

Answer 26

Joshua Lederberg
Lac z minus mutants - didn’t produce active beta-galactosidase
Lac Y minus mutants - unable to use lactose as an energy source

Question 27

Polycistronic mRNA:

Answer 27

is an mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs.

Question 28

Polycistronic mRNA: of the lac operon

Answer 28

Nederberg mapped all these genes
- order Z-Y-A
- they are transcribed as a single polycistronic mRNA = Co-ordinated
expression of all 3 genes

Question 29

How does lactose regulate transcription of the 3 structural genes?

Answer 29

1. Gene activity repressed in absence of lactose
   > Lactose induces expression
2. Lactose analog - IPTG (Isopropylthiogalactoside)
   (gratuitous inducer)
   = showed that induction is not mediated through interactions with
   enzymes
   - Because ITPG cant interact with the Beta-galactoside enzyme
3. Constitutive mutations
   - continuously turn the operon on, regardless if lactose was present
   or not.
   - mapping of the LacI mutation showed that the mutation was in a
   sight near to, but distinct from the structural genes
   = discovery of LacI gene = repressor gene (turns operon off)
   - LacO^c (second constitutive mutant) due to mutation in the operator
   region of the operon
   In both of these mutations:
   – lacI- and lacOc genes are produced continually and inducibility
   cannot happen - enzyme regulatability is lost

Question 30

Operon model – negative control

Answer 30

• lacI encodes an allosteric repressor molecule
  = a molecule that combined with another molecule (lactose) induces
  a conformational shift in the repressor and changes it chemical
  activity
• Repressor protein encoded by lacI binds to operator region
  > Inhibits RNA polymerase binding (prevents transcription)
• This represses the expression of the structural genes as the
  promotor can’t read through towards the structural genes
• Transcription thus only occurs when the repressor fails to bind to
  the operator = under negative control
• HOWEVER, when lactose is present, it can bind to the repressor
• This induces an allosteric conformational change - which changes
  the binding site of the lac repressor and prevents it from binding to
  the lac operator

Question 31

Functioning of the lac operon

Answer 31

Listen to slide 16 and 17

Question 32

Mutations in LacI and LacO^c

Answer 32

• Constitutive = continuous expression of lac operon
• The mutations interfere with molecular interactions between the lac
  repressor and the operator region

Mutations in lacI
= LacI-
- Either cause a conformational change - prevents it from binding to
the operator region
- or causes the complete loss of the repressor
> In both cases, it loses its ability to bind to the operator region,
which results in the operon being continuously on

Mutations in lacOc
- Alters the nucleotide sequence of the operator region
= normal repressor protein can no longer recognize that sequence
and is unable to bind to the operator

Question 33

Genetic proof for the operon model.
The operon hypothesis resulted in 3 major predictions which could be tested to determine the validity of the model

Answer 33

Hypothesis:
1. lacI produces a diffusible (trans-acting) product
2. lacO has no gene product - cis-acting element - involved in
regulating gene expression
> Also true for LacP - the promotor region
3. lacO must be adjacent to the structural genes in order to regulate
them, as they are a cis-element
> (also true for P)

How is this tested?
- bacteria may contain plasmids (in addition to their chromosome) which are independently replicated circular DNA molecules, that contain extra genes
> they have their own origins of replication = can replicate
independently of the bacterial chromosome

Listen to 20 - 28