Helene's Notes from reading

Beckwith, J. R., Zipser, D., The Lactose Operon, Cold Spring Harbor, 1970.

• Four transport systems with partially overlapping specificities for galactose and alpha-and beta-galactosides have been distinguished in E. coli. The first of these to be discovered was the lac system, which can defined as the transport system of which the product of the y gene is an integral part. A second system transports TMG but has little affinity for lactose or ONPG. It has been designated TMG permease II. A third transport system functions effectively in the concentration of D-galactose and methyl-1-thio-beta-D-galactopyranoside (TMG)This permease has been called the methyl-galactoside or MG permease. The fourth transport system appears in cells after induction by growth in the presence of D-fucose. D-galactose and and L-arabinose appear to be the only sugars of a number which have been tested which are effectiveky transported by this system, which has been termed the galactose permease. pp 51-52

• If the E. coli cells are grown on glucose as carbon source, the activity of the lac permease (and of beta-galactoside) is reduced as the result of catabolite repression, and in addition glucose or alpha-methylglucoside become powerful inhibitors of lac transports. p 67

THE LACTOSE REPRESSOR

• The binding of a substance to the region of the DNA molecules that that serves as an initiation point for the synthesis of a mRNA could block or affect or modify the synthesis of that messenger. The binding of a control substance to the mRNA could also affect the attachment of ribosomes or the synthesis of proteins.
• Operator: region on a DNA molecule to which a repressor binds. p 93

• specific regulatory genes: separate specific genes involed in the control of other genes. The product of a regulatory gene is a controlling substance. It acts as an intermediate to make a connection between a small molecule- a compound acting as a signal- and the target for the control. if the controlling gene makes a gene products that functions as a repressor, in the absence or suppression of the gene control, the structural gene runs at full rate.--> negative control.
• Positive control: The regulatory gene product is necessary for the DNA expression. If such control is exerted at the DNA level, a positive control gene might make a factor that binds to the RNA polymerase and permits that enzyme to initiate on a new region of DNA, or make a factor that binds to DNA , to open it for reading, or act negatively to prevent a stopping of the reading of DNA by interfering with an RNA stop signal. p 94

• If the cell is to respond to a chemical signal coming from outside, a number of steps will intervene before that signal affects the synthesis of roteins. The chemical signal must penetrate, be concentrated, possibly be altered, possibly trigger the release of other chemicals which, in turn, serve as signals- each step requiring a specific enzyme or function and so requiring the product of a specific gene.
• An operon is the set of the three structural genes z, y and a, and is thought of as being under the control of an operator. z, the gene for beta-galactose, the enzyme that splits lactose into glucose and galactose; y the gene producing a protein that is involved in the permeation (and active concentration) of lactose; and a, the gene for the thiogalactoside transacetylase, an enzyme which has no known in vivo function, strains deleted for the a gene behaving normally in all tests.
• There are three controlling elements: the i gene, p, the promoter, and o, the operator. The i gene makes a controlling substance, a repressor. The basic defining muattion in the igene is the change to i- : when no product is made by the i gene, if it were deleted, for example, then the structural genes function at full rate at all times; while in the wild-type (i+) cell, the enzymes are made at only 1000th of the full rate in the absence of sugar.
• Jacob and Monod theory states that the i gene makes some product that acts through the cytoplasm to prevent the expression of the lactose genes. the i- mutations are recessive constitutives, ie. the enzymes are made without being induced by the sugar, and the distribution of these i- mutations define the i gene. p 95
• The two other controlling regions do not appear to make any product, and are both defined through mutations that have only cis effects ie. changing the behavior only of the piece of DNA bearing the mutation.
• The promoter is defined genetically by mutations that prevent the expression of all three structural genes simultaneously. These mutations, p- occur outside the structural genes and affect only the physically adjacent genes in the cis position. The promoter region provides the starting site of the RNA for the operon, read out as one piece, and thus that the p- mutations change the rate of initiating RNA synthesis, for example, by being base changes that change the affinity of the RNA polymerase for this special region.
• How does the sugar induce? The hypothesis of Jacob and Monod states that the sugar , or an analog of the sugar, would prevent the attachment of the repressor to the operator, and so leave the genes open to function. The inducer might bind to the repressor in order to make it physically unable to interact with the operator. The sugar lactose itself is not an inducer. The sugar must be acted upon by beta-galactosidase, which as a transgalatoside transfers the galactose to some receptor to make the inducer. Thus the basal level of beta-galactosidase is needed to provide the first molecules of inducer. The permease must be functional to keep a high enough level of lactose in the cell for the induction by the sugar to be maintained. pp 96-97
• The lac repressor binds specifically to the lac operator DNA. The lac repressor binds only to DNA carrying the lac region.
• What does the inducer, IPTG, do to the binding? IPTG (isopropyl-thio-galactoside, unmetabolized inducer that cannot be split by beta-galactoside) prevents the binding. If the repressor is first bound to the DNA, then the inducer, IPTG, will cause the complex to fall apart. The inducer weakens the binding to the operator, the repressor comes off, and the operaon can function. p 100

TRANSCRIPTION OF THE LACTOSE OPERON IN E. COLI

• Transcription of the i gene is oriented in the same direction as the transcription of the 5 other genetic elements (p o z y a) which constitute the lac operon. p 119
• intermediary substances (catabolites), the production of which accompanies the metabolic conversion of energy-yielding compounds, act as corepressors for many inducible systems. This is the phenomenon of catabolite repression of which the glucose effect is a special case. Mutation within the promoter site of the lac regioncould generate complete insensitivityto catabolite repression. Catabolite repression inhibits initiation of messenger RNA synthesis.
• E. coli cells grown on glucose synthesise less 3'-5' cAMP than those maintained in a nonglucose containing medium. cAMP restores the normal level of lac RNA in cells induced in the presence of glucose. Presence of glucose reduces induction of lac RNA, but this effect is in great part overcome by cAMP. Neither glucose, nor cAMP do affect decay rate of lac RNA.

TRANSCRIPTION STARTS AND STOPS IN THE LAC OPERON

• Promoter: starting point for transcription. p 173

Neidhart, F. et al., Physiology of the bacterial cell, sinauer Associates, Inc., 1990.

• Enzyme induction is defined as an increased differential rate of synthesis of an enzyme (the rate of synthesis of the enzyme expressed relative to the rate of total protein synthesis) when some compound, the inducer, is added to the medium.
• Work on beta-galactosidase induction reached a climax when Jacob and Monod presented the operon model in 1961. Induction somehow releases the genetic message contained in the lac genes and makes it available at the sites where the new enzyme molecules are to be synthesized. It has been shown that mRNA appears and disappears quickly, as the kinetics of induction demands and that the quantity of the specific messenger present in the cells paralleled the rate of synthesis of beta-galactosidase. pp 331-332
• Two other proteins are invariably induced together with beta-galactosidase: galactoside permease (product of lacY) is a transmembrane protein involved in actively concentrating beta-galactosides within the cell by secondary active transport; galactoside acetylase (product of lacA) transfers acetyl groups to certain galactosides, presumably to reduce their inherent toxicity to E. coli. p 332

• A typical and very simple technique by which lac constitutive mutants (that is, mutants producing beta-galactosidase in the absence of inducer) have been isolated consists of spreading large numbers of wild-type E. coli cells on agar plates with phenyl-beta-D-galactoside as the only carbon source. p 334
• The lac repressor molecules are normally present in small numbers (about eight per cell). p 335
• The lac operon is indeed a small DNA segment.
• At points along a sequence of approximately 20 DNA base pairs(6-7 nm in length) the repressor makes contacts that result in firm and specific binding. p 336
• Unless the CAP (catabolite for activator protein), also called CRP (for cAMP receptor protein) is bound, transcription initiation from the lac promoter is weak. p 337
• Full expression of lac depends not only on the presence of a specific galactoside inducer, but also on the binding of the activator. The binding of CAP to DNA is itself dependent on the presence of a small nucleotide ligand, cyclic AMP (cAMP), which is required to hold CAP (an allosteric protein) in a suitable conformation for the binding. The requirement for CAP-cAMP for lac expression places this operon under an additional control: not only mnust a galactoside be present, but there must be a high intracellular level of cAMP as well. As a result, the lac operon belongs to a larger regulatory unit, the CATABOLITE REPRESSION NETWORK.
• The "natural" inducer of the operon is allolactose', not lactose. This isomer of lactose is produced as a side reaction to the hydrolysis of lactose by beta-galactosidase; The samll basal level of this enzyme in uninduced cells is sufficient to account for the generation of allolactose internally. The binding of cAMP-CAP complex facilitates effective binding of polymerase to the primary site (lac promoter site) but also blocks binding at the secondary site, so it stimulates transcription in two ways. p 338

Watson et al., Molecular Biology of the gene, The benjamin/Cummings Publishing Company, Inc., California, 1965,1987.

• cAMP is formed from ATP by the enzyme adenylate cyclase, which is bound to the cytoplasmic side of plasma membranes.
• cAMP works by stimulating the activity of protein kinases, enzymes that transfer terminal phosphate groups from ATP to specific amino acids on target proteins.
• RNA polymerases are the enzymes that transcribe RNA off DNA templates. These enzymes function only in the presence of DNA.
• beta-galactosidase is a bacterial enzyme which breaks the lactose into glucose and galactose.In the absence of lactose, the bacterium E. coli produces very small number of the molecules enzyme beta-galactosidase. In the presence of lactose, the bacterium produces a large number (thousands) of such molecules.

• A cell of E.coli has about 3000 molecules of RNA polymerase.