Genetics 8

Earths city lights Microbial Genetics 8Bacterial Gene Regulation(Operon Systems)

Constitutive Enzymes not controlled by induction or repression- produced by the cell under all physiological conditions, continuously all the time, because of their vital role in maintenance of cellular processes and structure essential to life always expressed by genes involved in vital biochemical processes= Examples: oxidoreductases catalyze oxidation-reduction reactions transferases transfer a functional group, e.g., methyl group lyases cleave bonds by means other than hydrolysis & oxidation isomerases catalyze isomerization changes within a single molecule ligases join two molecules with covalent bonds- produced only under conditions of advantage to cell, e.g., when cell is exposed to substances (substrates) upon which its enzymes act to form needed products Inducible (adaptive) Enzymes= Examples: -galactosidase hydrolyzes lactose to component monosaccharides penicillinase breaks down antibiotic penicillin as to make bacterium resistant controlled by induction or repression - induction only in presence of inducer substrate (e.g., lactose), and repression in presence of regulatory protein functioning as repressor in absence of inducer gene expression (transcription), thus, can be switched on and offConstitutive & Inducible Enzymes

- Operons can be inducible or repressible Genes grouped together and transcribed into a single mRNA molecule containing coding sequences for co-expression of more than one functionally related enzymes allows for coordinated control of genes required for metabolism; one switch controls more than one gene mRNA is dicistronic to polycistronic inducible products are made when the substrate is in the environment and needs to be metabolized (catabolic) repressible products are made when environmental signal molecule is scarce (anabolic) - Originally believed present only in bacteria and archaeaOperon - A setof two or more adjacent cistrons (genes) whose transcription is under the coordinated controlof a promoter, an operator, and a regulator gene now known also in bacteriophages (T7, T4, Lambda phage), eukaryotes in yeasts (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica), filamentous fungi (Cercospora, Aspergillus),insects (e.g., Drosophila), nematodes (e.g., Caenorhabditis elegans ), tunicates (e.g., Ciona intestinalis , Oikopleura dioica), protozoa (e.g.,Trypanosoma brucei), plants (e.g.,tomato)

F. Jacob and J. Monods 1961 Classic Operon Systems in Bacteria

Lac I (Regulatory gene) = encodes repressor protein which regulates synthesis of structural genes in operon located upstream, either adjacent to or distant from promoter site of operon active repressor binds to specific nucleotide sequence in operator region and thus, blocks binding of RNA polymerase (RNApol) to promoter and inhibits transcription lac repressor active in presence and inactive in absence of lactose O (Operator) = specific sequence on DNA to which an active repressor bindslac (Lactose) = the inducer molecule when lactose binds to repressor protein, repressor is inactivated; the operon is derepressed; and transcription (lac mRNA) of the genes for lactose utilization occursLac Z, Y, A = structural genes in lac operon Lac Z codes for -galactosidase, intracellular enzyme to cleave lactose; Lac Y, for lactose permease, a membrane-bound protein that pumps lactose into the cell; Lac A, for transacetylase that transfers an acetyl group from acetyl-CoA to -galactosides divided into two regions - (a) an upstream region, the CAP site. CAP (Catabolite Activator Protein) site is involved in catabolite repression of lac operon, and (b) a downstream region, the interaction site for RNApol. If repressor protein binds to the operator, RNApol is prevented from binding with the promoter and initiating transcription of enzymes for lactose utilizationFunctional & Regulatory Components of Lac OperonP (Promoter) = specific sequence on DNA to which RNA polymerase binds to initiate transcription

similar to lactose, but component monosaccharides linked 1-6 instead of 1-4 via transglycosylation of lactose by -galactosidasetransglycosylation an inducer of lac operon, i.e., it binds to repressor, the latter then undergoes conformational changes as to cause its dissociation from lac operator in E. coli without repressor on operator, the RNA polymerase can proceed to transcribe mRNA lac operon Lactose Allolactose(Enzymatic Breakdown & Transglycosylation)

Genes Turned On (Positive Control) Lactose (allolactose) is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter, and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase Genes Turned Off (Negative Control) There is no lactose to inhibit the repressor, so the repressor binds to the operator, which obstructs the RNA polymerase from binding to the promoter and carry out transcription of lac mRNA to make enzymes for lactose utilizationRepression & Induction of Lac Operon(Simplified)Repressed(without lactose)(no transcription)Active(with lactose)(transcription)

Regulatoryactive site (binds to promoter inthe absence of inducer)allosteric site(if with bound inducer causeschange in shape of repressor, which, in turn, can not bind to promoter) Details: Repression & Induction of Lac Operonlac genes are present but always turned off as long as substrate lactose is not present in the environment still continue to be off (repressor protein fixed in operator & blocking lac mRNA) as long as glucose is available to bacterium as carbon sourcepresence of lactose (inducer substrate is actually allolactose) renders the repressor regulatory protein inactive and unable to bind to operator lac RNA polymerase then is able to transcribe lac mRNA for translation to enzymes for lactosetranscription1 mRNA (for 3 structural genes)note change in shape ofregulatory (repressor ) protein

E. coli can use either glucose, a monosaccharide,or lactose, a disaccharide

However, lactose needs to be hydrolyzed (digested) first

So the bacterium prefers to use glucose when it canglucoselactoseAdaptation to Environmentwhere to ???

(1) When glucose is present and lactose is absent - E. coli does not produce -galactosidase (2) When glucose is present and lactose is present - E. coli does not produce -galactosidase (3) When glucose is absent and lactose is absent - E. coli does not produce -galactosidase (4) When glucose is absent and lactose is present - E. coli does produce -galactosidase Four Possible Situations

repressor protein blocks the promoter site where RNA polymerase must bind to if it were to transcribe the structural genes into mRNA for translation into enzymes for lactose assimilation regulatory gene lac I continuously synthesizes repressor protein (i.e., repressor protein is constitutive) repressor protein occupies operator site, a sequence of DNA just in front of the structural genes (lacZ, lacY, lacA) coding for enzymes in lactose metabolism Glucose Present, Lactose Absent

small amount of a sugar allolactose (an inducer) is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) binding of inducer allolactose causes the repressor protein to change its shape (a conformational change), thus, the latter can no longer occupy operator site RNA polymerase can now reach and bind to its promoter site, and then transcribe mRNA for the structural genes transcription of structural genesmRNALactose Present, Glucose Absent situations above explain in part how the lac operon IS transcribed only when lactose is present, but NOT why operon IS NOT transcribed when both lactose & glucose are present

when glucose and lactose are present, RNA polymerase is not blocked by inactive repressor protein (allolactose bound to allosteric site) and can occupy the promoter site, but it is unstable and it keeps falling off RNApolymeraseinactiverepressor protein a catabolite activator protein (CAP; CRP in some literature) must be present and it must bind to CAP site (C) to stabilize lac RNA polymerase in P site before transcription and subsequent translation (polysomal) still, RNA polymerase can not transcribe lac mRNA for the structural genesNO transcription of mRNAcataboliteactivator protein(inactive) the level of glucose is inversely proportional to that of cAMP because glucose inhibits the level of adenylate cyclase, the enzyme that converts ATP to cAMP Lactose Present, Glucose Present(Why Still No Transcription of Lac mRNA) CAP, by itself , is inactive. Its activation and binding to C site to stabilize RNA polymerase requires its change in conformational by binding to it of adenosine monophosphate (cAMP)

Positive control Positive Control in lac Operonbinding between CAP-cAMP complex in C site and RNA polymerase in P site helps unwind the DNA, and facilitates, stabilizes and assures maximal transcription and translation of genes Lac, Lac P, and Lac a binds to CAP site just before lac promoter sitetranscription of Lac mRNA(enzyme converts ATP to cyclic AMP. Its activity is inhibited when glucose concentration is high in the cell)

when glucose is absent, cAMP level is high, and substrate lactose is present to inactivate repressor protein. cAMP binds to CAP = CRP) cAMP-CAP complex then binds to the lac promoter and turns it on by helping RNApol to bind to the promoter Lactose Present, Glucose Absent, cAMP Level High- presence of lactose alone to inactivate repressor protein is not enough to deactivate and cause transcription of the operon, and the more so when glucose is present that leads to little or no cyclic adenosine monophosphate (cAMP) to activate catabolite repressor protein (CAP=CRP) glucose must also be absent since a cell will preferentially metabolize glucose over lactoseLactose Present, Glucose Present, cAMP Level Low

Summary Situations

- enzyme lactose permease (from gene lac y) is shut down when glucose is being transported into and metabolized by the cell This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxieDiauxieGlucose Effect (both glucose & lactose are present)glucoselactoselag

repressor is inactive if tryptophan (trp), itself the end-product amino acid of an active trp operon, is absentTryptophan Operon repressor is activated by presence of tryp functioning as co-repressor by binding to allosteric site of repressor for repressible operon to be transcribed, protein repressor must be in inactive form absence of trp means that the bacterium has to synthesize this amino acid from precursor molecules (NH4+ & a carbon source), e.g., when bacterium is in the guts of an organism that has ingested food with trp repressor and co-repressor in combination then occupies operator site to block RNApol from transcribing 5 structural genes coding for tryp synthesis transcriptiontranslationchange in shape of repressor protein when activated by tryp no transcription oftryp mRNA

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