Chapter 14

These molecules can also inhibit transcription in two ways

(1) Corepressor: small molecule that binds to a repressor protein, causing the protein to bind DNA (2) Inhibitor: Binds to an activator protein and prevents the protein from binding to DNA (3) Genes they regulate are termed repressible genes

Translation and Post-translational regulation

Refers to the functional control of proteins that are already present in the cell rather then regulation of transcription or translation.

Transcriptional regulation involves the action of two main types of regulatory proteins

(1) Repressor → Bind to DNA and inhibit transcription (2) Activator → Bind to DNA and increase transcription (a) Regulation via activator protein is termed positive control ii) Small effecter molecules effect transcription (1) Inducer: effecter molecule that speeds up transcription via two ways (a) Bind to a repressor protein, prevent the protein to bind DNA (b) Bind to a activator protein, causing the protein to bind DNA (c) Genes that are regulated in this way are termed inducible genes

Transcriptional Regulation

Most common way to regulate gene expression in bacteria by influencing the initiation of transcription

Regulation can occur at any point on the pathway to gene expression

Transcriptional level: whether or not to make the protein Translational: can bind to message and prevent the process (physical blockage in RNA) Post-translational

Repressor Proteins and Antisense RNA can inhibit translation

i) A transitional regulatory protein recognizes sequences within the mRNA, in most cases translational proteins act to inhibit translation, these are termed translational repressors. When translational binds to mRNA it can inhibit translational initiation in two ways: (1) It can bind in the vicinity of the shine Dalgarno sequence and/or start codon and thereby block the ribosomes ability to initiate translation in this region. (2) A second way is to regulate translation via the synthesis of antisense RNA (a) An RNA strand that is complementary to a strand of mRNA.

The LacI Gene Encodes a Diffusible Repressor Protein (EXP 14A)

i) Alternate hypothesis to explain how lacI- mutation could cause constitutive expression of the lac operon: correct explanation is the lac- mutation eliminates the function of the lac repressor protein, which prevents the protein from inhibiting the lac operon ii) Jacob, Monod and Pardee incorrect in their hypothesis that the mutation would result in the synthesis of an internal inducer that turns on the lac operon.

The trp operon is regulated by a repressor protein and also by attenuation

i) Attenuation can occur in bacteria because transcription and translation are coupled ii) Bacterial ribosomes quickly attach to the 5' end of mRNA soon after it's synthesis begins via RNA polymerase. During attenuation transcription begins but is terminated before the whole mRNA is made

Constitutive Genes

i) Constant levels of expressions c) Important for cellular processes such as i) Metabolism ii) Response to environmental stress iii) Cell division

Further Studies Have Revealed That the lac Operon Has _____ operator sites for the lack repressor

three

Jacob and Monod focused on lactose in E. coli. Key observations:

(1) The exposure of bacterial cells to lactose increased the levels of lactose utilizing enzymes by 1,000 to 10,000 (2) Antibody labeling techniques revealed that the increase in the activity of these enzymes was due to the increased synthesis of the enzymes. (3) The removal of lactose from the environment caused an abrupt termination in the synthesis of the enzymes (4) Mutations that prevented the synthesis of a particular protein involved in lactose utilization showed that a separate gene encoded each protein iii) These observations indicated to Jacob and Monod that enzyme adaptation is due to the synthesis of specific cellular proteins in response to lactose in the environment.

The Regulation of the Lac Operon Allows a Bacterium to Respond to Environmental Change

i) Small levels of proteins encoded by genes within the lac operon are continuously made, this along with the conditions where lactose in available in the environment and a leaky cytoplasm, allow for an increased concentration of lactose being transported into the cells. This causes the repressor protein un-bind lacO, enabling transcription of the lac Operon. When lactose is depleted from the environment, the sugars are metabolized and concentration levels lower, which then allowed the repressor protein to bind to the operator site, shutting of transcription

The lac Operon Is regulated by a Repressor Protein

i) The action of a small effector molecule is called allosteric regulation, allosteric proteins have at least two binding sites. The effector molecule binds to the proteins allosteric site, which is a site other then the two active sites. ii) Mechanism of induction of the lac operon (1) In the absence of the inducer allolactase, the repressor protein binds to the lacO site preventing RNA polymerase from transcribing the operon. (2) When alloctase is available in the environment it binds to the repressor protein altering the conformation of the repressor, which inhibits the repressors ability to bind to the operator site, this in turn allows RNA polymerase to transcribe the lac operon.

The lac Operon Encodes Proteins Involved in Lactose Metabolism

i) In Bacteria genes form together in a operon: group of two or more genes under the transcriptional control of a single promoter. An operon encodes a polycitronic RNA - RNA that contains the sequences for two or more genes. (1) Operons occur in bacteria because it provides the advantage of controlling all the genes involved in a common functional goal; the expression of the genes occurs as a single unit (Ex: the use of one light switch for multiple lights, instead of a single light switch for each light). ii) For transcription to occur an operon is flanked by a promoter - that signals the beginning of transcription - and a terminator - that specifies the end of transcription (two or more genes are between these two = operon). iii) Two distinctive transcriptional genes are present in lactose utilization and transcriptional regulation (1) Lac operon: contains a CAP site, promoter (lac P); operator site (lac O); three structural genes, LacZ, lacY, and lacA; and a terminator. (a) LacZ encodes the enzyme B-glactosidase (cleaves lactose into galactose and glucose). Also converts lactose into allallolactose, which acts as a effector molecule that regulates the lac Operon. (b) LacY gene encodes lactose permease, a membrane protein required for the active transport of lactose into the cytoplasm of the bacterium. (c) LacA gene encodes glactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs. Which may prevent toxic buildup within the bacterial cytoplasm. (d) CAP site - short DNA segments that function in gene regulation - CAP site is recognized by an activator protein called the Catabolic Activator Protein. (e) Operator site - short segment of DNA sequence that function in gene regulation - is a sequence of bases that provides a binding site for a repressor protein. (2) Second transcriptional unit involved in genetic regulation is the lacI gene (not apart of lac operon) (a) Is constitutively expressed at low levels, has it's own promoter (i). The lacI encodes lac repressor - protein important for regulation of lac operon. (i) Functions as homotetramere (four sub-units) (ii) Only small amounts needed to repress the lac operon

Inducible Operons encode catabolic enzymes and repressible operon

anabolic enzymes.

b) Posttranslational regulation can occur via feedback inhibition and covalent modifications

i) A common mech to regulate the activity of metabolic enzymes is feedback inhibition (1) Convert precursor molecules to a particular product, then the final product in a metabolic pathway then inhibits an enzyme that acts early in the pathway ii) Allosteric enzyme contains two different binding sites (1) The allosteric site binds the final product of the metabolic pathway. When bound to the regulatory site, the final site inhibits the catalytic ability of enzyme one.

The Phenomenon of Enzyme Adaptation Is Due to the Synthesis of Cellular Proteins

i) Enzyme adaptation refers to the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme. When a bacterium is not exposed to a substrate, it does not make the enzymes needed to metabolize that substance.