Gene Expression in Prokaryotes

1. Gene Expression in Prokaryotes

2. Why regulate gene expression? • It takes a lot of energy to make RNA and protein. • Therefore some genes active all the time because their products are in constant demand. • Others are turned off most of the time and are only switched on when their products are needed.

3. The control of gene expression is much more complex in eukaryotes than in prokaryotes. • Reasons being, Eukaryotes have: • Compartmentalization of cells • More extensive transcript processing • Regulation from a distance • Cell and tissue specific gene expression • Larger Genome size • Genes scattered about the genome

4. In prokaryotes, the control of transcriptional initiation is the major point of regulation • In eukaryotes the regulation of gene expression is controlled nearly equivalently from many different points: • Initiation of transcription (most important control) • Chromatin control • Epigenetic control • Transcript processing • Transcript stability • RNA transport • Protein stability • Protein transport • Post-Translational modifications

5. Gene Control in Prokaryotes • One way in which prokaryotes control gene expression is to group functionally related genes together so that they can be regulated together. • This grouping is called an operon. • The clustered genes are transcribed together from one promoter giving a polycistronic messenger.

6. Gene Control in Prokaryotes • An operon can be defined as a cluster gene that encode the proteins necessary to perform coordinated function • Genes of the same operon have related functions within the cell and are turned on (expressed) and off together (suppressed). • The first operon discovered was the lac operon so named because its products are involved in lactose breakdown.

7. An operon consists of: • a promoter (binding site for RNA polymerase) • a repressor binding site called an operator that overlaps the promoter. • structural genes

8. Operator • Repressor proteins encoded by repressor genes, are synthesized to regulate gene expression. • They bind to the operator site to block transcription by RNA polymerase. Promoter • The promoter sequences are recognized by RNA polymerase. • When RNA polymerase binds to the promoter, transcription occurs

9. Actvators • The activity of RNA polymerase is also regulated by interaction with accessory proteins called activators • The presence of the activator removes repression and transcription occurs

10. Two major modes of transcriptional regulation function in bacteria (E. coli) to control the expression of operons: • repression and • induction. • Both mechanisms involve repressor proteins. • Induction happens in operons that produce gene products needed for the utilization of energy. • Repression regulates operons that produce gene products necessary for the synthesis of small biomolecules such as amino acids.

11. Inducible system Negative control the effector molecule interacts with the repressor protein such that it cannot bind to the operator • With inducible systems, the binding of the effector molecule to the repressor: • greatly reduces the affinity of the repressor for the operator • the repressor is released and transcription proceeds.

12. In addition to negative control mediated by a repressor, expression from an inducible operon is also under positive control, mediated by an activator • A classic example of an inducible (catabolite-mediated) operon is the lac operon, responsible for obtaining energy from galactosides such as lactose.

13. Repressible system Negative control the effector molecule interacts with the repressor protein such that it can bind to the operator • With repressible systems, the binding of the effector molecule to the repressor: • greatly increases the affinity of repressor for the operator • the repressor binds and stops transcription. • For the trp operon , the addition of tryptophan (the effector molecule) to the E. coli environment shuts off the system because the repressors binds at the operator.

14. In addition to negative control mediated by a repressor, expression from a repressible operons is attenuated by sequences within the transcribed RNA. • A classic example of a repressible (and attenuated) operon is the trp operon, responsiblefor the biosynthesis of tryptophan.

15. Structure of the lac Operon • The lac operon three structural genes: • Z • y • a • The z gene codes for β-galactosidase , responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose.

16. The y gene codes for permease, which increases permeability of the cell to galactosides. • The a gene encodes a transacetylase. In addition to the structural genes the lac operon also has regulatory genes: • Promoter: Binding site for RNA polymerase • Operator: Binding site of repressor

19. Control of lac operon expression • The control of the lac operon occurs by both positive and negative control mechanisms. Negative control of the lac operon What happens to lac operon when glucose is present and lactose is absent? • During normal growth on a glucose-based medium (lacking lactose), the lac repressor is bound to the operator region of the lac operon, preventing transcription.

20. What happens when glucose is absent and lactose is present? • The few molecules of lac operon enzymes present will produce a few molecules of allolactose from lactose. • Allolactose is the inducer of the lac operon. • The inducer binds to the repressor causing a conformational shift that causes the repressor to release the operator.

21. With the repressor removed, the RNA polymerase can now bind the promoter and transcribe the operon.

22. Positive Control of the lac operon What happens when both glucose and lactose levels are high? • Since the inducer is present, the lac operon will be transcribed. • However the rate of transcription is very slow (almost repressed) because glucose levels are high and therefore cAMP levels are low.

23. The repression of the lac operon under these conditions is termed catabolite repression and is as a result of the low levels of cAMP that results from an adequate glucose supply. • This repression is maintained until the glucose supply is exhausted. What happens when glucose levels start dropping in the presence of lactose? • As the level of glucose in the medium falls, the level of cAMP increases. • Simultaneously the inducer (allolactose) is also binding to the lac repressor (since lactose is present).

24. The net result is an increase in transcription from the operon. • The ability of cAMP to activate (increase) expression from the lac operon results from an interaction of cAMP with a protein termed CRP (for cAMP receptor protein). • The protein is also called CAP (for catabolite activator protein).

25. The cAMP-CAP complex binds to a region of the lac operon just upstream of the promoter • The binding of the cAMP-CRP complex to the lac operon stimulates RNA polymerase activity 20-to-50-fold. • (Repression of the lac operon is relieved in the presence of glucose if excess cAMP is added.) • cAMP is therefore an activator of the lac operon. • This type of regulation by an activator is positive in contrast to the negative control exerted by repressors.

27. trp operon • The trp operon encodes the genes for the synthesis of tryptophan. • As with all operons, the trp operon consists of the promoter, operator and the structural genes. • It is also subject to negative control by a repressor • In this system, unlike the lac operon, the gene for the repressor is not adjacent to the promoter, but rather is located in another part of the E. coli genome. • Another difference is that the operator resides entirely within the promoter • Unlike an inducible system, the repressible operon is usually turned on.

28. Structure of the trp operon The operon consists of: • The operon consists of 5 structural genes that code for the three enzymes required to convert chorismic acid into tryptophan’ • The operon also contains a gene coding for a short oligopeptide (trpL) which functions in attenuation • Operator • promoter

29. Gene Gene Function P/O Promoter; operator sequence is found in the promoter trp L Leader sequence; containing attenuator (A) sequence the leader trp E Gene for anthranilate synthetase subunit trpD Gene for anthranilate synthetase subunit trpC Gene for glycerolphosphate synthetase trp B Gene for tryptophan synthetase subunit trp A Gene for tryptophan synthetase subunit

30. Negative control of trp operon • The affinity of the trp repressor for binding the operator region is enhanced when it binds tryptophan, blocking further transcription of the operon and, as a result, the synthesis of the three enzymes will decline. • hence tryptophan is a corepressor. • This means that when tryptophan is absent expression of the trp operon occurs • the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell.

32. Attenuation of the trp operon • Expression of trp operon is reduced by the addition of trytophan in trpR mutants. • Further research established that this second level of tryptophan control involved two components: 1. tRNA, specifically tryptophanyl-tRNA, tRNATrp, i.e. tRNATrp charged with tryptophan. 2. the trpL gene  

33. The attenuator region is composed of sequences found within the transcribed RNA of the operon • It is involved in controlling transcription from the operon after RNA polymerase has initiated synthesis of the proteins. • The leader sequences are located prior to the start of the coding region for the first gene of the operon (the trpE gene).

34. The leader sequence (trp L) contains tandem tryptophan codons. How does this affect transcription of the trp operon? • It contains two consecutive trp codons and therefore serves to measure the tryptophan supply in the cell. • If the supply is good, then the tRNA will be charged and the leader peptide will be translated without problem. • If the supply is inadequate, then the tRNA will not be charged, and translation will stall at the trp codons.

35. The trpL mRNA region can adopt a number of different conformations. It contains several self-complementary regions which can form a variety of stem-loop structures

36. Different stem-loops can form: • Depending on the level of tryptophan in the cell and hence the level of charged trp-tRNAs • Depending on the position of ribosomes on the leader polypeptide and • Depending on the rate at which they are translated

37. trp Operon Transcription Under High Levels of Tryptophan • Recall that transcription and translation can occur simultaneously in bacteria. • This means that the ribosome will attach to mRNA and is able to influence the formation of secondary structures by the mRNA.

38. In the case of the trpL mRNA, when the cellular levels of tryptophan are high, the levels of the tryptophan tRNA are also high. • Immediately after transcription, the ribosome follows right behind RNA polymerase until it is halted by a stop codon. • Translation is quick because of the high levels of tryptophan tRNA. • This permits formation of the terminator stem-loop which will cause RNA polymerase to dissociate (recall rho independent termination of transcription in prokaryotes)

39. How is the terminator stem-loop formed? • Because of the quick translation of domain 1, domain 2 becomes associated with the ribosome complex. • Then domain 3 binds with domain 4, and transcription is attenuated because of this stem loop formation. • The stem loop formed by binding of domains 3 and 4 is found near a region rich in uracil and acts as the transcriptional terminator loop (see transcription notes from unit 3) • Consequently, RNA polymerase is dislodged from the template.

40. trp Operon Transcription Under Low Levels of Tryptophan • Under low cellular levels of tryptophan, the translation of the short peptide on domain 1 is slow. • As a result domain 2 does not become associated with the ribosome. • Rather domain 2 of the leader mRNA associates with domain 3 of the leader mRNA. • This step loop structure is the anti-terminator. Its formation prevents formation of the terminator

41. This structure permits the continued transcription of the operon. Then the trpE-A genes are translated, and the biosynthesis of tryptophan occurs • Domain 4 is called the attenuator because its presence is required to reduce (attenuate) mRNA transcription in the presence of high levels of tryptophan. • Domain 1 is also an important component of the attenuation process. • The section of the leader sequence encodes a 14 amino acid peptide that has two tryptophan residues.

43. References • http://www.biocourse.com/ui/swf/iLabs/lac_operon.swf • http://faculty.plattsburgh.edu/donald.slish/Att-Trp.html • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation28.html