BOIL 366 Reg_Gen_Exp_bacteria_lac__ara__etc

Graded control of the trp operon through transcription attenuation.

(a) Structure of the mRNA from the trp operon •The mRNA generated from the trp operon includes a 5' leader sequence containing four regulatory regions labeled 1 - 4. •"Sequence 1" is translated into the leader peptide, which regulates the trp operon. •The leader peptide has 14 aa, two of which are Trp. •When both free, sequences 2 and 3 can base pair to make a stem-loop structure. •3 and 4 can base-pair to form a G≡C-rich stem-and-loop (hairpin) structure which acts as a transcriptional terminator.

Regulation of the ara operon

(a)When arabinose is absent (and glucose is present, i.e. cAMP is low): • AraC protein forms a dimer in which one monomer binds to araO2 and the other to araI1, forming a DNA loop •This prevents RNA polymerase from binding and transcription of the ara operon.

Regulation of the gal operon.

(b) The Gal repressor: • Does not prevent RNA Pol to bind the promoter. •Prevents promoter-Pol OPEN complex formation, required for transcription initiation.

Graded control of the trp operon through transcription attenuation.

(b) When [tryptophan] is high, [tRNATrp ] is also high. Thus ribosomes translate quickly through Trp codons of sequence 1 and into sequence 2. This allows sequences 3 and 4 to form a hairpin that stalls the RNA polymerase and terminates transcription.

Regulation of the ara operon.

(b) When arabinose is present (and glucose is not, in which case cAMP is high) •AraC binds arabinose, and then activates the ara operon. •The AraC dimer changes conformation such that one monomer binds araI1 and the other binds araI2. Binding of AraC to araI2 recruits RNA polymerase to the promoter and activates transcription of the ara operon.

Negative regulation of the lac operon by the Lac repressor. Fig 20-4 When lactose is not present:

-Lac repressor binds the operator region -This prevents RNA Pol from binding the promoter site and transcribe the operon. Note: Expression from LacI is constitutive.

Regulation of the lac operon by CRP.

-The cAMP levels increase in response to low glucose -CRP-cAMP to bind the reg. region -RNA Pol robustly binds and transcription from the lac operon is much stronger

Regulation of the lac operon by CRP.

-The repressor dissociates from the operator. -However, low cAMP levels prevent CRP-cAMP formation and DNA binding. -Transcription from the lac operon is week.

Regulation via catabolite repression: A positive regulatory system.

Glucose is metabolized directly by glycolysis and is E. coli's preferred energy source. When glucose is available, a regulatory mechanism known as catabolite repression restricts expression of the genes required for metabolizing other sugars (e.g., lactose, arabinose, etc.) even when these sugars are also present. Regulation via catabolite repression: A positive regulatory system. In the absence of glucose cell's increase the expression of genes that allow the use of alternative food sources.

Positive regulation of the lac operon by CRP.

In the absence of lactose the repressor binds the operator, blocking RNA polymerase and preventing transcription of the lac genes This is regardless of glucose availability and, thus, binding of CRP-cAMP to the operon.

Chemical structures of some effectors of the lac operon.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) can also bind the Lac repressor and cause its dissociation from the operator, inducing transcription of the lac operon.\ IPTG is not a substrate for β-galactosidase. It is an "inducer" of the operon. The β-galactoside 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) serve as a substrate for β-galactosidase, producing a blue color when metabolized. X-gal does not induce expression of the lac operon.

The Lac operon encodes for three proteins that mediate lactose metabolism in E. coli.

LacY: A membrane protein (Galactoside permease, encoded by lacY) allows entry of lactose into the cell. LacZ: Lactose is converted to galactose and glucose, and a small amount of allolactose, the inducer of lac operon by β-Galactosidase (encoded by lacZ) Lac A: A thiogalactoside transacetylase protein encoded by lacA gene. It modifies toxic galactosides that are imported along with lactose, facilitating their removal from cells.

Coordinated Transcription of several genes: The SOS system

Notes: •Autocleavage of LexA repressor requires RecA protein. •DNA damage creates sites of single-stranded DNA, which are quickly bound by RecA protein. DNA-bound RecA becomes a coprotease for LexA, and their association facilitates the destruction of LexA and induction of the SOS response.

Interaction of the Lac repressor and the Lac operatorn.

The Lac repressor is a tetramer formed from two homodimers. Each homodimer can bind one operator sequence. -The lac operon contains three operator sequences O1 to O3. •Only O1 is adjacent to the lac operon promoter. •To repress the operon, one dimer binds to the O1 and the other dimer binds simultaneously to one of O2 or O3. The simultaneous binding of the Lac repressor to O1 and to O2 or O3 results in a looped DNA structure, providing an effective steric block to transcription.

The lactose (lac) operon of E. coli.

The lac operon includes three genes All three genes are transcribed as a single unit from the same promoter. The operator region regulates transcription through interaction with a repressor protein, encoded by lacI. The repressor: - has a separate promoter (i.e, is transcribed separately) - is constitutively expressed.

Regulation of the Trp Operon

The trp operon encodes five genes that control synthesis of tryptophan The trp operon is regulated by a repressor system and attenuation. (a)In the absence of tryptophan the Trp repressor cannot bind the operator, and transcription from the trp operon is initiated. (b) When tryptophan is abundant, expression from the trp operon is not needed. •Tryptophan serves as the effector molecule for the Trp repressor. •Association of tryptophan and repressor causes the repressor to bind the operator, blocking transcription.

When lactose is present,

its metabolite allolactose binds the Lac repressor, inducing conformational changes that causes the repressor to dissociate from the operator. RNA polymerase can then initiate transcription.

Blue-white selection using pUC vectors

•Following cloning, vectors that took in the DNA of interest must be selected. Sometimes the DNA is not inserted due to: i.Contamination with the undigested vector ii.Vector re-ligating to itself. The blue-white selection technology allows selecting for bacteria that harbor the vector WITH insert. Transformed bacteria are grown on medium containing IPTG and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). And amp. •Bacteria harboring pUC vector containing the insert DNA remain white. Why? •Bacteria harboring the empty pUC vector (i.e., no insert DNA) turn blue. Why?

Coordinated Transcription of several genes: The SOS system.

•In the default (normal; no damage) state of the E. coli cell, the LexA repressor prevents transcription of the SOS genes. •In response to DNA damage, LexA undergoes autocleavage, inactivating itself and allowing transcription of the SOS genes.

(c) Like the lac and ara operons, the gal operon:

•Is transcribed only when glucose is absent. •Prevents promoter-Pol OPEN complex formation, required for transcription initiation. •Thus, binding of CRP-cAMP is required.

Regulation via catabolite repression: A positive regulatory system.

•The effect of glucose on expression of the lac genes is mediated by two elements: Cyclic AMP (cAMP), a small-molecule effector The activator cAMP receptor protein, or CRP, a homodimer which can bind both DNA and cAMP simultaneously. •When glucose is absent, CRP-cAMP binds to a site near the Lac promoter and stimulates RNA transcription fiftyfold. •CRP-cAMP is thus a positive regulatory factor responsive to glucose levels. •This is unlike the Lac repressor, which is a negative regulatory factor responsive to lactose. •The positive and negative regulatory systems act in concert.

Coordinated Transcription of several genes: The SOS system.

•The expression of a number of genes involved in repair of damaged DNA in bacteria is induced in a coordinated manner. This is known as the SOS response. •Genes involved in the SOS response are located at different sites in the chromosome. •The SOS response requires two key regulatory proteins: •The RecA protein •LexA repressor protein

(c) In the absence of tryptophan:

•Trp tRNA is low, and ribosomes stall on Trp codons of sequence 1, allowing sequences 2 and 3 to associate to make a hairpin. •With sequence 3 unavailable to associate with sequence 4, the terminator structure is not formed and transcription can proceed. •The amount of free tryptophan available for protein synthesis thus determines whether the trp operon is transcribed.