Prokaryotic Gene Regulation:

1. Prokaryotic Gene Regulation: Lecture 5

2. 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]

3. 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.

4. 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.

5. Regulatory loops: transcription Gene • 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 product

6. 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…

7. 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 “lacoperon” must be expressed. • In order to ensure efficiency the “lacoperon”: • 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 lacoperon: • 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

8. 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

9. Function of Lac operon Repressor protein • 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? RNA polymerase Klug chapter 15

10. Glucose and the lacoperon • 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

11. 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

12. 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

13. 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]