DAIRY BIOTECHNOLOGY

Module 2. Fundamental biological principles

Lesson 7
GENE REGULATION - THE ‘LAC OPERON’

7.1 Introduction

All the metabolic functions in a cell with regard to both degradative and biosynthetic pathways in prokaryotes and eukaryotes are performed through the expression of specific genes encoded on their DNA for growth and development. However, all the genes present in a cell are not expressed all the time unless their functions are absolutely indispensable. Majority of the genes are turned on only when the products of such genes are needed for the growth in a given environment (signal). Their expression is turned off, when their products are either no longer needed or cell already has adequate amounts of these products. This switching on and off of the gene expression in a cell constitutes a very powerful tightly regulated system for controlling gene functionality. By turning off the expression of genes when their products are not required, an organism can avoid unnecessary wasting of energy since the energy resources available in a cell are limited. The cell has to utilize the conserved energy resources very judiciously to synthesize products that maximize the cellular growth rate. Gene expression in a bacterial population is primarily regulated by three mechanisms i.e. constitutive, inducible and repressible systems operating in these organisms.

7.2 Constitutive Expression

Constitutive gene expression is not regulated which is typical of genes whose products are indispensable for cellular functions. These genes always remain turned on and keep producing the desired enzymes or proteins continuously and stably all the time for the growth and survival of the cell. Such genes are called house keeping or constitutive genes that encode RNA and proteins having basal vital functions such as rRNA, ribosomal proteins and glycolytic/respiratory enzymes.

7.3 Inducible and Repressible Genes

Inducible and repressible gene products are required only under certain circumstances. Inducible genes are ‘turned on’ in response to the presence of a substrate (inducer) in the environment e.g. lactose in lac operon.

Repressible genes are ‘turned off’ in response to an environmental signal e.g. the simultaneous presence of lactose/glucose or xylose/glucose which will repress the genes required for utilization of lactose or xylose.

7.4 Negative and Positive Regulation of Gene Expression

Bacteria alter the gene expression by using positive or negative regulation. Fundamental difference between positive and negative regulation is when the regulatory molecule i.e. repressor alone (lac operon) or repressor along with inducer/end product (‘trp operon’) is binding to the promoter and also whether the molecule is increasing (inducer) or decreasing (repressor) the gene expression.

Prokaryotic gene expression is tightly regulated at the level of transcription, translation and enzyme functions. Since transcription and translation are coupled in prokaryotes, the level of control in gene expression is relatively higher at transcriptional level.

7.5 What is an Operon?

An operon constitutes a genetic switch operating in prokaryotes only to coordinately regulate a cluster of genes involved in metabolic pathways (degradative and biosynthetic) for their functionality. These genes are transcribed together under the control of the same promoter into a single polycistronic mRNA which is eventually translated into their individual polypeptides. Operon consists of three basic elements : The structural genes, the regulatory sequences viz. promoter and operator regions and the regulatory gene. The best known example of the operon system is the ‘lac operon’ in E. coli which is now used extensively as a model for understanding the mechanism of lactose utilization in bacteria.

7.6 Lac Operon in E. coli

An operon is a functional unit of gene expression in bacteria. This represents the unique gene regulatory system operating in prokaryotes only. The ‘lac operon’ model was proposed by Jacob and Monod in 1961 to describe coordinated regulation of genes encoding enzymes required for utilization of lactose in E. coli.

7.6.1 Elements of ‘lac operon’

The ‘lac operon’ consists of the following key elements as shown in Fig.7.1.

7.6.1.1 Three structural genes

‘Lac operon’ is comprised of three structural genes viz. lacZ, lacY and lacA encoding enzymes/proteins that are functionally related for bringing about the metabolism of lactose in E. coli. All these three genes are under the control of a single promoter and are involved in the breakdown of lactose.

i) LacZ codes for β-galactosidase which breaks down lactose into glucose and galactose.

ii) LacY codes for lactose permease required for the transport of lactose into E. coli cells.

iii) LacA codes for thiogalactoside transacetylase whose precise function is not known as yet.

7.6.1.2 Regulatory sequences

The regulatory sequences of the ‘lac operon’ include the following.

i) LacO - the operator region (O) at which the ‘lac’ repressor binds to block the promoter for RNA polymerase binding, thereby, turning off the ‘lac operon’.

ii) LacP - the promotor region (P) at which RNA polymerase binds to initiate the transcription of the structural genes into polycistronic mRNA.

iii) LacI - the regulatory gene which encodes the trans acting ‘lac’ repressor protein that binds at the operator region to block the transcription in the absence of lactose –the inducer of lac operon. In fact, the real inducer of the lac operon is the allo-lactose an isomer of lactose.

iv) LacI is not located within the lac operon but upstream several nucleotides away from the lac operon along with its own promoter to synthesize the ‘lac’ repressor

Apart from these, there are some effector molecules which either activate or deactivate the binding of the repressor or RNA polymerase to their respective sites at the promoter-operator regions of the lac operon. The organization of the ‘lac operon’ and its functioning in the regulation of the structural genes required for lactose metabolism in E. coli are illustrated schematically below

7.7 Working of Lac Operon

The lac operon is regulated differently in presence or absence of the inducer i.e. lactose (allolactose) as described below

7.7.1 In absence of lactose

When lactose is absent from the growth medium inoculated with E. coli, lac repressor is synthesized by expressing lacI from its promoter with the help of RNA polymerase in active form and diffused into the medium to reach the operator site in the operon. The binding of the repressor at the operator blocks the binding of RNA polymerase at the promoter and thus the structural genes are turned off and hence the polycistronic mRNA is not synthesized by the operon. The regulation of ‘lac operon’ in absence of inducer i.e. lactose is shown in Fig. 7.2.

7.7.2 In presence of lactose

When lactose is available in the growth medium, it will bind with the repressor at its inducer binding site and alters the conformation of the repressor which can no longer bind with the operator site on the lac operon. Hence, there is no hindrance in the binding of the RNA polymerase at the promoter and therefore, all the three structural genes are transcribed into a polycistronic mRNA from which all the three enzymes required for lactose metabolism are translated independently to perform their specific functions. The regulation of ‘lac operon’ in the presence of inducer i.e. lactose is shown in Fig. 7.3.

In fact, the real inducer of lac operon is not lactose but its isomer allolactose which is produced from lactose with the help of β-galactosidase. However, the major limitation of lactose (Allolactose) serving as the inducer when added in the medium is its constant utilization by E. coli during growth and hence needs to be replenished continuously to keep the induction of lac operon get going. This problem has by and large been solved with the synthesis of IPTG (Iso Propyl Thio Galactoside) a structural analogue of lactose -a gratuitous inducer of the lac operon which acts as a an efficient artificial inducer without being metabolized by the bacterium and hence remains there in the growth medium indefinitely. IPTG is extensively used in the laboratory in inducing the expression of heterologous genes in E. coli for large scale production of high value recombinant proteins.

7.7.3 Lac operon is under both negative and positive regulation

Lac operon is a classical example of an operon which is subject to both negative and positive regulation. The negative regulation as already mentioned is mediated by the repressor molecule which will bind the operator in the absence of the inducer and thus will turn the structural genes off by inhibiting their transcription. The positive regulation of lac operon is triggered by a signal molecule called cAMP (Cyclic Adenosine monophosphate) which senses the depletion of glucose concentration in the medium (When E .coli cells are under starvation conditions). Under high glucose concentration in the medium, the level of cAMP in the cell goes down and conversely, at low glucose concentrations, cAMP level in the cell increases. cAMP activates another protein called CRP (cAMP Receptor Protein) also designated as CAP (Catabolite Activator Protein) which will bind at the CRP site in the lac operon located near the lac promoter only when it is complexed with cAMP. The binding of the active CRP will strengthen the binding of RNA polymerase at the promoter, thereby, increasing the expression of lac operon considerably. The working of positive regulation of ‘lac operon’ has been demonstrated in Fig. 7.4.

Further Reading

Books

Fundamental Bacterial Genetics, Nancy Trun, Janine Trempy (Eds), Wiley-Blackwell, 2003, ISBN: 978-0-632-04448-1

From Genes to Genomes: Concepts and Applications of DNA Technology, 3^rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6

Microbial Genetics, 2^nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Internet Resources

http://en.wikipedia.org/wiki/Lac_operon

biosiva.50webs.org/Lacoperon.htm

http://www.blackwellpublishing.com/trun/pdfs/Chapter12.pdf