Control of Gene Expression in Prokaryotes

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.

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 RNA).

Gene Control in Prokaryotes The prokaryotic genes organized in to operons. 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 (suppressed) together. The first operon discovered was the lac operon so named because its products are involved in lactose breakdown.

An operon consists of: Promoter: binding site for RNA polymerase. Operator: binding site of repressor, overlaps the promoter. Structural genes

Promoter The promoter sequences are recognized by RNA polymerase, When RNA polymerase binds to the promoter, transcription occurs. 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.

Activators 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.

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

Inducible system Also called Positive 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 as a result the repressor is released and transcription proceeds. A classic example of an inducible operon (catabolite-mediated) is the lac operon, responsible for obtaining energy from galactosides such as lactose.

Repressible system Also called 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. A classic example of a repressible (and attenuated) operon is the trp operon, responsible for the biosynthesis of tryptophan.

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

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.

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

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 but the rate of transcription is very slow (almost repressed) because glucose levels are high and therefore cAMP levels are low. The repression of the lac operon under these conditions is termed catabolite repression and is 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 binds to the lac repressor (since lactose is present). 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).

cAMP is therefore an activator of the lac operon. The cAMP-CAP complex binds to a region of the lac operon just upstream of the promoter. This binding 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.

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.

Structure of the trp operon The operon consists of: Five structural genes that code for the three enzymes required to convert chorismic acid into tryptophan. A gene (trpL) which functions in attenuation. Operator promoter

trp L Leader sequence; containing attenuator (A) sequence the leader 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 trp D : Gene for anthranilate synthetase subunit trp C: Gene for glycerolphosphate synthetase trp B: Gene for tryptophan synthetase subunit trp A: Gene for tryptophan synthetase subunit

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 co-repressor, 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.

References: Brock Biology of microorganisms, 2012. Molecular Biology of the Cell, Bruce Alberts, 2008. Molecular cell biology, Darnell, Lodish, and Baltimore 2008. Review Articles: Regulation of RNA polymerase I transcription in the nucleolus - Genes and Develop., 2003. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond - FASEB J., 2001. Translational Control of Viral Gene Expression in Eukaryotes - Microbiology and Molecular Biology Reviews, 2000.