Lac Operon

helix-turn-helix

1. A recognition helix of 7-9 amino acids contacts the bases in the major groove. These contacts are specific. 2. The amino acids of the helix make hydrogen bonds with particular bases in the DNA. 3. A sharp turn connects the recognition helix to a second helix, that makes non-specific contacts with the DNA. These help position the recognition helix in the major groove. How the lac repressor binds DNA

lac Operon is also inducible by CAP-cAMP.

1. CAP will only bind to the CAP binding site if it is also bound to cAMP. So, if glucose is low, cAMP is high, CAP binds, and the operon is induced. If glucose is high, cAMP is low, CAP doesn't bind, and transcription of the operon is low. This is how the information about glucose level is transmitted. This system is an example of positive regulation, and the operon is again inducible.

lac operon is inducible

1. Inducible by lactose since there is always a low level of transcription of the lac operon 2. If lactose is present, some of it enters the cell, where some of it is isomerized to allolactose as a side reaction of β-galactosidase. The allolactose binds to lac repressor. This causes a conformational change of the protein, which prevents it from binding the operator. With the repressor out of the way, RNA polymerase can transcribe the structural genes. Thus, allolactose is the effector for this system of inducible gene regulation.

Low trp levels

1. No attenuation 2. If tryptophan levels are low, then the concentration of tRNA-trp is also low. 3. When the ribosome reaches the trp codons, it stalls, waiting for the tRNA-trp that is not available. 4. Having the ribosome sitting there prevents the region1-region2 loop from forming. Instead, the region 2- region 3 loop forms. 5. This loop is not followed by a string of Us, that sequence is further away to the 3' side. This structure of the RNA does not cause termination, and transcription continues.

lacl gene

1. encodes the repressor 2. it is not part of the operon. 3. It has its own promoter and terminator, and its transcript is separate from the transcript encoding the structural genes.

trpL

1. first part of the polycistronic RNA encoding all the enzymes needed for tryptophan biosynthesis 2. When there is little tryptophan in the cell, transcription proceeds beyond trpL and produces the full-length transcript. 3. When tryptophan is abundant, synthesis of the transcript terminates at the end of the sequence encoding the leader peptide. This early termination of transcription is called attenuation.

lac operon is negatively regulated by

1. lac repressor forms a dimer that binds to the operator sequence in the DNA 2. with repressor, RNA polymerase can't proceed along the DNA and produce the transcript --> negative regulation

regulation of trp operon

1. negative, repressible regulation 2. in absence of repressor and tryptophan, operon is ON 3. trp repressor without tryptophan cannot bind to operator 4. In presence of tryptophan, tryptophan binds to trp repressor protein, changing conformation so it can bind to operator and turn operon OFF

Attenuation

1. second mechanism affecting control of the trp operon 2. can work in bacteria because transcription and translation are coupled. 3. As soon as an mRNA appears outside RNA polymerase, ribosomes can assemble on it and begin translation, allowing a system for control of operons producing enzymes of amino acid biosynthesis.

RNA sequence of trp attenuator

1. three stretches of RNA that can participate in formation of stem-loop structures 2. not all the stems can form at once 3. region 3-4 stem is followed by a string of Us 4. In the absence of a ribosome translating trpL, the nature of the hairpins formed is determined by the order in which they are synthesized. Region 1 rapidly hybridizes with region 2. Therefore, region 2 doesn't bind with region 3. Instead, region 3 hybridizes with region 4. This creates an intrinsic terminator, and transcription stops. no trp- regions 2 and 3 bind, 3 and 4 dont so no attenuation with trp, 3 and 4 bind, causing attenuation

lacA

Encodes transacetylase Covalently modifies lactose and analogues

Conditions

Lactose is present, glucose is absent glucose is more efficient than lactose no lactose, no need to make enzymes

three operator sites of lac operon

binding to O1 site has some repressing effect, but less than others Binding to O1 and O2, or O1 and O3 is almost as good as the complete operon More than one binding site is needed because tetramer formation between dimers bound to different sites creates loops in the DNA, blocking transcription more effectively

lacZ

encodes B-galactosidase β-galactosidase also converts lactose into allolactose (an isomer that is the inducer of the operon) Cleaves lactose and lactose analogues

lacY

encodes lactose permease membrane protein required for transport of lactose and analogues into the cell

trp operon

genes should be expressed when the cell is short of tryptophan, but not when there is plenty of tryptophan available. 5 structural genes: trpE, trpD, trpC, trpB, trpA 2 regulatory genes: trpR (repressor protein), trpL(leader peptide)

cAMP

high when glucose is low binds to CAP

allolactose

inducer for lac operon

CAP

must bind to cAMP to activate transcription of the lac operon by RNA polymerase.

Inducible vs repressible regulation

1. Operons involved in catabolism (breakdown of a substance) are typically inducible --> the substance to be broken down (or a related compound) acts as the inducer 2. Operons involved in anabolism (biosynthesis of a substance) are typically repressible --> The inhibitor or co-repressor is the small molecule that is the product of the operon

High trp levels

1. attenuation occurs 2. If the levels of tRNA-trp are high, the ribosome does not pause at the trp codons. Instead, it completes the leader peptide and pauses at the stop codon of trpL. 3. In this position, the region3 - region 4 stem forms, which is the terminator structure as it is followed by a string of Us and Transcription terminates.

Lac operon is positively regulated by

1. controls the operon according to the presence or absence of glucose. 2. cAMP is inhibited by glucose so, when glucose is high, cAMP is low, and when glucose is low, cAMP is high. This is the system for sensing the level of glucose. 3. Since the promoter is weak in the absence of a repressor, CAP binds to the promoter and to RNA polymerase, helping the polymerase bind to the promoter

lac operon summary

1. negatively regulated by lac repressor ■ Repressor: product of lacI gene ■ Small effector molecule: allolactose, an inducer ■ Allolactose levels increase in presence of lactose positively regulated by CAP-cAMP ■ Activator: CAP-cAMP ■ Small effector molecule: cAMP, an inducer ■ cAMP levels decrease in the presence of glucose