Prokaryotic Gene Regulation:

Lecture 5

overview

• Generic types of regulation control

• Regulation of the “sugar” lactose gene(s) for the bactria e. coli [ referred to as the lac operon]

• Regulation of the expression of the “amino acid” gene tryptophan in E. Coli. [try operon]

Gene Regulation• All “genes” must have some way of regulating their expression in order to allow them

to adopt appropriately to the environment.

• In prokaryotic cells the process, owing to the simple nature of the genomic material, is controlled mainly at the transcription level…

• Essentially the molecule “RNA polymerase” must bind to the “exposed “ DNA strand; it must then move, in the 5’ to 3’ direction, transcribing “all” the DNA of the gene

• The transcription is controlled/ regulated at: – RNA polymerase binds to the DNA;

• RNA binds DNA transcription begins • RNA is prevented from binding; DNA is not transcribed: gene not expressed.

– RNA polymerase moves in the 5’ to 3’ direction:• If it is prevented from moving: transcription is stopped : gene not expressed• Otherwise transcription is completed and the gene is expressed.

•

Classes of transcriptional control

• Inducible: gene is expressed only if the molecule (inducer) is present.

• Repressible: if molecule is present gene expression is turned off

• Negative control: gene expression occurs unless it is switched off.

• Positive control: gene is “off” unless it is switched on.

Regulatory loops: transcription • Feedback loop: product of

the gene expression loops back (directly/indirectly) and alter the expression of the same gene (lactose).

• The effect can be either :– positive : increases the level

of transcription [lac ]– Negative: decreases the

level of transcription [tryp]

Gene

Gene product

Sample gene regulatory systems

• Lactose “gene” system is “turned on” by its Inducer: (lactose)

• Tryptophan “gene” system is “turned off” by its repressor: (tryptophan)

• Alternatively they can be described as Feedback loops under:– Negative control: expression has to be turned “off” – Positive control: expression must be turned on…

Prokaryotic regulation: lactose• Lactose, a complex sugar (glucose)

• In order for E. Coli to use (metabolise) the sugar a gene system referred to as the “lac operon” must be expressed.

• In order to ensure efficiency the “lac operon”:– will not be expressed if there is no lactose– will be expressed if there is lactose.

• However, Glucose, a more efficient energy source, alters this function of The lac operon:– Will not be expressed if glusose is present– Will not be expressed if no glucose and no lactose– Will be expressed if no glucose but there is lactose

Function of Lac operon

• The term operon is the common “gene system” used in prokaryotic cells and generally a number of genes are regulated as a one.

• In the E Coli Lac operon there are:– 1 repressor gene (lacI) and 1 repressor protein– 3 structural genes: LacZ, LacY and LacA– A Cis–acting regulatory region (operator)– A promoter (where RNA polymerase binds)– A leader region (not critical to expression)– The operon is a positive controlled

Klug chapter 15

Function of Lac operon• RNA polymerase binds to the promoter region• The repressor gene produces a product “a

repressor protein”• This binds to the DNA at the operator region

and blocks RNA polymerase moving down the DNA strand.

• If lactose is present it alters the repressor protein.

• The alter repressor protein is unable to bind to the DNA

• RNA polymerase binds to the promoter region and begins transcribing the 3 structural genes.

• When lactose levels drop to zero: what happens?

Klug chapter 15

RNA polymerase

Repressor protein

Glucose and the lac operon • Lactose is metabolised into

glucose so what happens if glucose is present.

• Catabolite-activation protein (CAP): CAP must be present to make RNA polymerase binding efficiently

• In the pressence of glusoce the CAP is altered and prevents RNA polymerase binding to the promoter region and so prevents transcription.

Klug chapter 15

The tryptophan operon• Tryptophan is an essential AA and is

normally made (biosynthesised) by E Coli. If tryptophan is absent in the growth medium

• If tryptophan is present in a growth medium then the biosynthesis stops because

• The repressor protein is altered by tryptophan and the modified repressor protein now binds to the operator region and blocks RNA polymerase transcribing the enzymes required to make tryptophan.

• This is an example of a repressor / repressible operon.

• What type of “control” is used by the tryp operon Klug chapter 15

The tryptophan operon • In addition in the presence of

tryptophan there is an additional control mechanism called:

• The attenuation regulatory mechanism:

• In the sequence prior to structural genes is the attenuator region:

• If tryptophan and its gene expression is repressed they still found that transcription was initiated… ; there was “RNA” fragments of leader [L]sequence

• Thus altering the repressor protein is not enough to prevent expression.

• It seems that tryptophan also binds to the attenuator [A]region and prevents transcription beyond the leader region.

Attenuator region

Leader region

Klug chapter 15

Exam question• Gene expression can be controlled both negatively and

positively. Explain using suitable examples how both forms of control are achieved in prokaryotic cells.

• Gene regulatory systems can be controlled via an inducer or repressor. Discuss the difference between both methods and illustrate your answer with suitable example

• Distinguish between the complete functionality of the tryp operon and the lac operon [include glucose/attenuation]