Bio3400 Chapter 16 Regulation of Gene Expression in Prokaryotes
  1. Prokaryotes respond to environmental conditions by regulating transcription via            ,               , or              control.
  2. In E. coli, lactose metabolism is regulated by           control of an            system, where          is the inducer.

      The E. coli lac operon contains 3 structural genes: lacZ, lacY, and lacA, as well as an upstream regulatory region consisting of an operator and a promoter (binding site for RNA polymerase).

      The structural genes of the lac operon are transcribed into a single mRNA, which is then translated into the three enzymes. The lacZ gene encodes b-galactosidase, an enzyme that digests the disaccharide lactose into glucose and galactose. The lacY gene codes for the enzyme permease, a transport protein which facilitates the diffusion of lactose into the cell. The lacA gene codes for the enzyme transacetylase.

      The enzyme b-galactosidase catalyzes the conversion of the disaccharide lactose into its monosaccharide units, glucose and galactose.

      The lacI gene exerts negative control over the operon by producing an allosteric repressor which can repress of the structural genes unless it is bound by the inducer, lactose.

      When the repressor binds to the operator region, it inhibits the RNA polymerase after the latter binds to the promoter, repressing the transcription of the structural genes. The operon is off.

      In a lac I^- mutant, the repressor protein is altered and cannot bind to the operator, so the structural genes of the operon are constitutive (always turned on).

      In a lac O^ mutant, the nucleotide sequence of the operator DNA is altered and will not bind with a normal repressor protein, so the structural genes are always turned on.

      When lactose is present, it indirectly induces the activation of the genes by binding with the repressor, which becomes inactive, and the operon is turned on.

      In a lac I^S mutant, the repressor cannot interact with the inducer, lactose. As a result, the repressor always binds to the operator, and the structural genes are permanently repressed.
    • Expression of the lac operon is also subject to positive control: the operon is             by the catabolite-activating protein (CAP), together with Cyclic adenosine monophosphate ( cAMP ) when          is absent.

        Glucose is a preferred sugar source for E. coli than lactose. In the absence of glucose, Cyclic adenosine monophosphate (cAMP) levels increase, resulting in the formation of a CAP-cAMP complex, which binds to the CAP site of the promoter, stimulating transcription. Continue

        In the presence of glucose, cAMP levels decrease, CAP-AMP complexes are not formed, and transcription is not stimulated. This is called catabolite repression.

        The enzyme adenyl cyclase catalyzes the formation of cAMP from ATP. The activity of adenyl cyclase is inhibited by glucose. Thus cAMP levels fall when glucose levels rise.
    • Binding of the repressor to the           forms a             loop in the lac operon DNA.

      The lac operon. The numbers represent nucleotide sites upstream and downstream from the initiation of transcription. Binding of the repressor to operators O[1] and O[3] creates a repression loop, which prevents access of RNA polymerase to the promoter.
  3. The E. coli tryptophan (trp) operon is a              system that is repressed by              of the transcript.

      In addition to the repressor, promoter, operator and structural genes, the tryptophan (trp) operon contains a leader and attenuator sequences.

      In the absence of tryptophan, the repressor is inactive and cannot bind to the operator , thus allowing transcription to proceed (constitutive).

      When tryptophan is present, it functions as a corepressor by binding to the repressor. This complex binds to the operator, leading to a partial repression of the operon by attenuation.

      At the 5' end of the trp operon mRNA is a leader peptide (LP) and an attenuator region (A); the LP contains UGG triplets which encode tryptophan. This sequence can be folded into either of two types of hairpin structures: a terminator or an antiterminator.

      When tryptophan is abundant, the ribosome proceeds normally through the UGG codons in the leader peptide (LP). As a result, a terminator hairpin forms, and mRNA synthesis is terminated near the end of the attenuator (A); this is called attenuation.

      If tryptophan is scarce, the ribosome stalls at the UGG codons in the leader peptide (LP). This induces the formation of the antiterminator hairpin within the transcript. As a result, attenuation does not occur, and transcription proceeds, leading to expression of the entire set of structural genes.
    • Bacillus subtilis uses a combination of       and AT proteins to regulate its trp operon via              .

      The trp RNA-binding attenuation protein (TRAP) of B. subtilis consists of 11 subunits, each capable of binding one molecule of tryptophan. When saturated with tryptophan, TRAP can bind to the 5'-leader sequence of the mRNA, forming a terminator hairpin and lead to attenuation of expression of the trp operon. If tryptophan is scarce, an anti-TRAP (AT) protein inhibits binding of TRAP to the leader sequence, stopping the attenuation.
  4. The arabinose (a monosaccharide) operon is subject to both positive and negative regulation by the       protein.

      In addition to structural genes, a catabolite-activating protein (CAP) gene, and Operator region, the ara operon contains a regulatory araC that encodes a regulatory protein that acts as either an inducer (in the presence of arabinose) or a repressor (in the absence of arabinose) by binding to the Inducer site.

      In the presence of arabinose, the araC protein binds only to the Inducer site, and the system is induced. Maximum expression occurs when cAMP levels are also high, forming the CAP-cAMP complex.

      In the absence of arabinose, the araC protein binds to both the Inducer site and the O[2] sites, forming a loop in the DNA and repressing transcription of the structural genes. Where is the promoter?
    • + The promoter should be in front of the operator.