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THE Lac OPERON and Trp Operon

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THE Lac OPERON and Trp Operon Central University of South Bihar Panchanpur, Gaya, India E-Learning Resources Department of Biotechnology NB: These materials are taken/borrowed/modified/compiled from various sources like research articles and freely available internet websites, and are meant to be used solely for the teaching purpose in a public university, and for serving the needs of specified educational programmes. Programme: M.Sc. Biotechnology; 2nd semester Molecular Biology and Genomics Course Instructor: Dr. Krishna Prakash THE lac OPERON and Trp operon © 2007 Paul Billiet ODWS The Operon system ◼ The control of protein synthesis has been elucidated in E.coli. ◼ Wild E.coli growing on minimal medium (glucose+nitrogen source) is able to synthesise all the amino acids need for its growth. ◼ This synthesis requires the presence of certain enzymes, which are produced by the growing E.coli cells. ◼ If the required amino acids are supplied in the culture medium, the cells stop synthesising these amino acids and enzymes required for the synthesis of these amino acids are not produced in the E.coli cells. © 2007 Paul Billiet ODWS ▪ This is called repression, and the enzymes whose synthesis is repressed are called repressible enzymes. ▪ For example, synthesis of arginine from glutamic acid involves three steps, each step catalyzed by a separate enzyme. The three steps are: Enzyme II Glutamic acid Enzyme I Ornithine citrulline Enzyme III arginine If arginine is absent--------all three enzymes synthesised If arginine is present---------no one enzyme synthesized If citrulline is added----------only enzyme III synthesized • If lactose is present ------------lactose degrading enzyme synthesis started • This is referred to as induction and enzymes produced are known as inducible enzymes • If lactose is absent then vice-versa step will take place • In E.coli this control of gene activity ie., repression or induction is operative at the transcriptional level and needs the presence of associated genetic controlling elements with the regulated genes Operons ◼ An operon is a group of genes that are transcribed at the same time. ◼ They usually control an important Jacob, Monod & Lwoff biochemical process. © NobelPrize.org ◼ They are only found in prokaryotes. © 2007 Paul Billiet ODWS Structure of operon Effector molecules bind to repressor and change its ability to interact with operator The lac Operon • The lac operon consists of three genes (lac z, y and a)each involved in processing the sugar lactose • One of them is the gene for the enzyme β- galactosidase • This enzyme hydrolyses lactose into glucose and galactose © 2007 Paul Billiet ODWS Structure and components of the lac-operon of E.coli © 2007 Paul Billiet ODWS The control of the lac operon © 2007 Paul Billiet ODWS Four situations are possible 1. When glucose is present and lactose is absent the E. coli does not produce β-galactosidase. 2. When glucose is present and lactose is present the E. coli does not produce β-galactosidase. 3. When glucose is absent and lactose is absent the E. coli does not produce β-galactosidase. 4. When glucose is absent and lactose is present the E. coli does produce β-galactosidase © 2007 Paul Billiet ODWS 1. When lactose is absent ◼ A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site ◼ The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing Repressor RNA protein Blocked polymerase DNA I O z y a Regulator Operator lac operon gene site © 2007 Paul Billiet ODWS 2. When lactose is present ◼ A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) ◼ This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site DNA I O z y a © 2007 Paul Billiet ODWS 2. When lactose is present ◼ A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) ◼ This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site DNA I O z y a Promotor site © 2007 Paul Billiet ODWS 3. When both glucose and lactose are present ◼ This explains how the lac operon is transcribed only when lactose is present. ◼ BUT….. this does not explain why the operon is not transcribed when both glucose and lactose are present. © 2007 Paul Billiet ODWS ◼ When glucose and lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off Repressor protein removed RNA polymerase DNA I O z y a Promotor site 4. When glucose is absent and lactose is present ◼ Another protein is needed, an activator protein. This stabilises RNA polymerase. ◼ The activator protein only works when glucose is absent ◼ In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose Activator protein stabilizes the RNA Transcription polymerase DNA I O z y a Promotor site © 2007 Paul Billiet ODWS CAP ( catabolize activator protein) ◼ A regulator gene crp present else where in the E.coli genome produces a regulator know as CAP. ◼ Also known as CRP (cyclic AMP receptor protein) ◼ CAP protein binds to controlling element near the lac promoter there by enhancing transcription (positive control) CAP continued…… ◼ The addition of glucose causes CAP protein to leave the controlling element and lac transcription slows down to very low level. ◼ Presence of glucose inhibits the production of lactose utilization enzymes, a phenomenon known as catabolite repression. ◼ This repression is brought about by two intermidiates molecules, CAP and cAMP ◼ In presence of cAMP, CAP stimulates the transcription of lac operon ◼ When glucose is added then the cAMP level goes down (reason unknown) and this lowers the activity of activator CAP, which results in drop in transcription of lac operon genes ◼ When glucose and lactose is present in medium , E. coli utilizes it as a carbon source over lactose. ◼ Thus, CAP function as a activator and cAMP as a effector molecule. ◼ Lac promoter in fact contains 1. RNA pol. Binding site and 2. CAP binding site. trp operon ◼ The trp operon codes for an enzymes needed for the synthesis of tryptophan ◼ This is an anabolic pathway as opposed to the catabolic pathway for the lac operon ◼ Bacterial cells should have the trp operon “ON” in the absence of tryptophan and “OFF” in the presence of tryptophan © 2007 Paul Billiet ODWS • The trp operon consists of a promoter, an operator, a leader (trp L), an attenuator, and 5 structural genes, designated trp E,trp D, trp C, trp B, and trp A. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Regulation of the E.coli trp operon • Trp R is a regulatory homodimer molecule • Each homodimer contains HTH motif which interact with half of the trp operon • Trytophan function as a molecular “on” or “off” switch for trp operon • Charles Yonofsky in 1972 showed the function of Trp R • In his experiment he made E.coli mutants that lack a functional Trp R protein showed increase in trp operon transcription after being starved for W. • It means Trp R function as a regulatory molecule for trp operon Reference Book • Cell Biology By Gerald Karp 7th Edition • The Cell: A Molecular Approach by Geoffrey M. Cooper.
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