Lecture 13 - Catabolite Repression of the lac Operon •...

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Unformatted text preview: Catabolite Repression of the lac Operon • When glucose is present, lac operon is in a relatively inactive state • Selection in favor of glucose attributed to role of a breakdown product, catabolite • Process known as catabolite repression uses a breakdown product to cause repression of the operon Positive Control of lac Operon • Positive control of lac operon by a substance sensing lack of glucose that responds by activating lac promoter – The concentration of nucleotide, cyclic-AMP, rises as the concentration of glucose drops Catabolite Activator Protein • cAMP added to E. coli can overcome catabolite repression of lac operon • Addition of cAMP lead to activation of the lac gene even in the presence of glucose • Positive controller of lac operon has 2 parts: – cAMP – Protein factor is known as: • Catabolite activator protein or CAP • Cyclic-AMP receptor protein or CRP • Gene encoding this protein is crp Proposed CAP-cAMP Activation of lac Transcription • The CAP-cAMP dimer binds to its target site on the DNA • The aCTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAP • Binding is strengthened between promoter and polymerase Strong induction of the lac promoter requires lactose and reduced glucose concentrations in the culture medium. Major Shifts in Bacterial Transcription Infection of E. coli by Phage l • Virulent phage replicate and kill their host by lysing or breaking it open • Temperate phage, such as l, infect cells but don’t necessarily kill • The temperate phage have 2 paths of reproduction – Lytic mode: infection progresses as in a virulent phage – Lysogenic mode: phage DNA is integrated into the host genome the phage genome Events Leading to Lysogeny - The Prototype Phage: Lambda Circularization of the phage chromosome - Lambda DNA is a double stranded linear molecule with small single stranded regions at the 5' ends. In the cell the free ends of the circle can be ligated to form a covalently closed circle as illustrated below. Site-specific recombination In phage lambda, the integration site is known as att P, in E. coli the site is att B. The integration reaction (att B x att P) is mediated by the proteins integrase (Int) and host integration factor (IHF). Integration: When integration occurs, two new sites are created, att L and att R, flanking the integrated prophage, with no loss of DNA sequence. ATT sites contain a 15-bp recognition sequence for the recombinase (integrase). Excision: When att L x att R recombine (mediated by the proteins integrase and host integration factor and excisionase [Xis]), the lambda -DNA is excised from the E. coli genome, recreating the att B site in E. coli and the att P site in lambda. Lysis and Lysogeny - The lambda phage is adsorbed on the lamB receptor. - only expressed when the bacteria are grown in the presence of maltose. 1. Lysis: multiplication. Most phages take over the machinery of the host cell to produce a large number of viral particles. 2. Lysogeny: rest. This silent phage can go lytic under certain conditions. - A lysogenic phage is called a prophage (integrated into the chromosome) - Lysogenic bacteria have immunity against further infection - The prophage can be induced and is excised from the bacterial genome The phage genome Lytic Reproduction of Phage l • Lytic reproduction cycle of phage l has 3 phases of transcription: – Immediate early – Delayed early – Late • Genes of these phases are arranged sequentially on the phage DNA One of 2 immediate early genes is cro – cro codes for a repressor of cI gene that allows lytic cycle to continue – Other immediate early gene is N coding for N, an antiterminator Antitermination • Antitermination is a type of transcriptional switch • A gene product serves as antiterminator that permits RNA polymerase to ignore terminators at the end of the immediate early genes • Same promoters are used for both immediate early and delayed early transcription • Late genes are transcribed when another antiterminator permits transcription of the late genes from the late promoter to continue without premature termination Lysis and lysogeny are controlled by the proteins encoded by cro and cI genes. The lambda phage will remain in the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if Cro proteins predominate cI genes transcription and translation will regulate lysis vs lysogeny The cI dimer may bind to any of three operators, OR1, OR2, and OR3, in the order OR1 > OR2 >OR3. Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2: OR1 and OR2 are almost always simultaneously occupied by cI dimer . (a) In the absence of cI proteins, the cro gene may be transcribed --- lysis (b) In the presence of cI proteins, only the cI gene may be transcribed --- lysogeny (c) At high concentration of cI, transcriptions of both genes are repressed --- lysogeny When the host DNA is damaged (e.g., under UV irradiation), the cI protein may be cleaved by certain protease promoted by the RecA protein. Lytic or Virulent Phages Modified lac Promoters • tac promoter Has (-)10 sequence of lac, (-)35 sequence of trp separated by 16 bp • trc promoter Has (-)10 of lac, (-)35 of trp separated by 17 bp trc and tac are 3X stronger than trp and 10X stronger than lac Phage T7 Promoter • Strong promoter • Requires T7 RNAP for transcription • T7 RNAP production can be transferred to chromosome or vector and placed under lac repressor control • High level of RNAP production allows high target gene expression ...
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This note was uploaded on 04/09/2011 for the course CHEM 4461 taught by Professor Max during the Spring '08 term at Lamar University.

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Lecture 13 - Catabolite Repression of the lac Operon •...

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