Saturday, May 8, 2010

Bacterial Transcription: Induction and The Lac Operon

A continuation from previous transcription posts: Regulation and Attenuation and Initiation, Elongation, and Termination.

Gene induction is a phenomenon that is incredibly fast, taking only two to three minutes for a cellular response.  During induction, the actual enzyme levels in the cell rise, and inhibitors of protein synthesis prevent induction (providing further evidence that it is, in fact, protein synthesis that is necessary for induction).  The lac operon is the most commonly studied gene that uses induction for regulation.

During initial studies of the lac operon, there were two types of genes considered: structural and regulatory genes.  Structural genes are those that encode the actual metabolic enzymes; regulatory genes are involved in controlling the expression of the structural genes.  The lac operon was convenient for study because it had an observable phenotype (the production of a gene in the presence of glucose or lactose) and because mutants could be generated that had different phenotypes.  After mutagenizing bacteria, the researchers screened E.coli on plates with glucose and X-galactose.  Colonies of bacteria that were inducible turned white and did not express β-galactosidase, and regulatory gene mutants would be able to express β-galactosidase in the absence of lactose (and turn blue).  Mapping of the genes that were responsible for these phenotypes led to the identification of the o and i regions.  Mutations in either of these regions resulted in a constitutive phenotype. 

Further analysis of the lac operon and induction led to the creation of a model:  the i region codes for the inducer, which binds the lac operon DNA in the promoter region (identified via DNA-binding assays and footprint analysis).  Later structural studies identified the i protein contains an HTH motif, as well as IPTG-binding domains.  The i gene, called the repressor can bind the promoter region of the lac operon and prevent RNA polymerase from binding.  With the presence of glucose but not lactose, the lac repressor binds the operator sequence of the genome and it prevents RNA polymerase and its helper protein CAP (bound to cAMP).  CAP-cAMP induces a bend in the DNA, which allows RNA polymerase to bind, and CAP has its own binding site the DNA that helps to position the polymerase.  With the presence of lactose, the repressor binds to the lactose and no longer binds the operator.  Therefore, RNA polymerase can bind the promoter sequence and promote transcription of the lac genes.  However, in the absence of glucose, which is indicative of a high concentration of cAMP (low ATP), CAP binds cAMP and promotes stronger interaction of polymerase and the lac promoter.   

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