CHAPTER 9 Molecular Mechanisms of Gene Regulation Gene regulation mechanisms that determine the types of cells in which a gene will be transcribed when and where it will start and end how it will be spliced duration of time 9 1 Regulation of transcription is a common mechanism in prokaryotes In bacteria and bacteriophage on off gene activity is often controlled through transcription When a gene product is needed transcription of the gene is turned ON OFF very little synthesis of the gene product Coordinate regulation control of synthesis of several proteins by a single regulatory element Prok proteins are usually translated from a single mRNA molecule Prok mRNA is polycistronic Euk mRNA is monocistronic In negative regulation the default state of transcription is on Negative regulation default ON transcription takes place until its turned OFF by repressor protein that binds to DNA upstream from start site either INDUCIBLE or REPRESSIBLE Inducible transcription Repressor DNA binding protein normally keeps transcription OFF presence of inducer molecule repressor binds with inducer and loses DNA binding allowing transcription to occur common in catabolic degradative pathways Repressible transcription default is ON until active repressor turns it OFF Regulatory protein is the aporepressor which has no DNA binding activity Active repressor aporepressor co repressor results in cessation of transcription common in anabolic biosynthetic pathways The final product is typically the co repressor In positive regulation the default state of transcription is off Binding with a regulatory protein is necessary to turn it ON The protein that turns transcription on is a transcriptional activator protein Prok negative regulation is more common Euk positive regulation is more common Some genes exhibit autoregulation which means that the protein product of a gene regulates its own transcription In negative autoregulation the protein inhibits transcription and high concentrations of protein result in less transcription of mRNA In positive autoregulation the protein stimulates transcription more protein made transcription increases to maximum rate weak induction can be amplified 9 2 In prokaryotes groups of adjacent genes are often transcribed as a single unit The first regulatory mutations that were discovered affected lactose metabolism Metabolism of lactose in E coli requires beta galactosidase which cleaves lactose to yield galactose and glucose and a transporter molecule lactose permease which is required for the entry of lactose into the cell F lac is a plasmid carrying the genes for lactose utilization The lacZ gene codes for beta galactosidase and the lacY gene for permease lacZ and lacY happen to be adjacent in the chromosome Lactose utilizing enzymes can be inducible regulated or constitutive Lac E coli in medium lacking lactose beta galactosidase beta galactosidase and permease concentrations are low If lactose is present numbers are 1000 fold higher If lactose is added to a Lac growing in lactose free and glucose free medium beta galactosidase and permease are synthesized simultaneously The addition of the lactose in this situation triggers synthesis of the lac mRNA Transcription of lactose genes is inducible transcription and lactose is a inducer of transcription IPTG isopropylthiogalactoside is a sulfur containing analog that induces but is not cleaved by beta galactosidase Constitutive mutants are produced in the absence as well as in the presence of an inducer TABLE ON P 297 Repressor shuts off messenger RNA synthesis The lacI gene is a regulatory gene whose product is the repressor protein that keeps the system turned off Because the repressor is necessary to shut off mRNA synthesis regulation by the repressor is negative regulation A lacI mutant lacks the repressor and transcription is constitutive The lacI s mutations are dominant and shut off mRNA synthesis from both F plasmid and chromosome whether or not the inducer is present Uninducible The lactose operator is an essential site for repression A lacO c mutation causes constitutive synthesis of beta galactosidase only when the lacO c and lacZ alleles are contained in the same DNA molecule The lacO c mutation is said to be cis dominant only genes in the cis configuration same DNA molecule are expressed in dominant lacO cannot alter constitutive active of lacO c mutation cannot be complemented The lacO region is called the operator which is a binding site in the DNA for repressor protein The lactose promoter is an essential site for transcription lacP mutations like lacO c mutations are cis dominant a wildtype lacZ remains inexpressible in the presence of a lacP Lac system lacI lacP lacO lacZ lacY lacP mutations cannot be complemented lacP promoter where RNA polymerase binds allowing initiation The lactose operon contains linked structural genes and regulatory sequences OPERON MODEL ONE the lactose utilization system consist of two kinds of components structural genes lacZ and lacY which encode proteins needed for transport and cleavage of lactose and regulatory elements repressor lacI promoter lacP and operator lacO TWO the products of the lacZ and lacY genes are coded by a single polycistronic mRNA lacA is the enzyme beta galactoside transacetylase lac operon lacZ lacY lacP and lacO THREE the promoter mutations lacP eliminate the ability to synthesize lac mRNA FOUR the product of lacI gene is a repressor which binds to operator DNA sequences FIVE when repressor is bound to operator initiation of transcription of lac mRNA by RNA polymerase is STOPPED SIX inducers stimulate mRNA synthesis by binding to and inactivating the repressor If inducer is present operator is not bound with repressor promoter available for mRNA initiation Binding with the repressor prevents transcription so the regulation of the operon requires the lacO operator either overlap or be adjacent to the promoter of the structural genes lacI is a soluble protein and is diffusible throughout the cell doesn t have to be near lacO The lactose operon is also subject to positive regulation If glucose and lactose are present in the growth medium lac operon transcription is shut down until all glucose is consumed Cyclic adenosine monophosphate cAMP mediates action of hormones conc indirectly regulated by glucose metabolism glucose cAMP low glycerol cAMP high lactose cAMP high Glucose levels regulate the cAMP concentration in the cell and cAMP regulates the activity of