Question

So, i have been doing some lactose mutant analyses (beta-galactosidase assay, and complementation test).

From the result obtained from the complementation test, a mutant has defection in lac Z gene where beta-galactosidase is produced. on the other hand, i also undertook the beta-galac assay. And, the results are really vague to interpret.

The first thing is I want to focus on is basal-level transcription. Wild type displays the 0.1 of beta-galactosidase activity due to basal-level transcription (when before the addition of IPTG). But the mutant also records around 0.1 in beta-galactosidase activity without IPTG.

if the lac Z is mutated, i guess, the figure should be really small or zero. But, still, it is able to produce the same amount of beta-galactosidase as wild type does.

how would you interpret?

any advice or suspected another mutation region in lac operon? (please be fully honest, if you not sure about your answer, let me know)

you dont need figures...(read carefully.)

Answer 1

The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose in E.coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize.

Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as a sugar source for the bacterium, the three genes of the lac operon can be expressed and their subsequent proteins translated: lacZ, lacY, and lacA. The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. lacY encodes Beta-galactoside permease, a membrane protein which becomes embedded in the cytoplasmic membrane to enable the cellular transport of lactose into the cell. Finally, lacA encodes Galactoside acetyltransferase.

Normally, the repressor searches for the operator by rapidly binding

and dissociating from nonoperator sequences. Even for sequences that mimic

the true operator, the dissociation time is only a few seconds or less. Therefore,

it is easy for the repressor to find new operators as new strands of DNA are

synthesized. However, when the affinity of the repressor for DNA and operator

is increased, it takes too long for the repressor to dissociate from sequences on

the chromosome that mimic the true operator, and as the cell divides and new

operators are synthesized, the repressor never quite finds all of them in time,

leading to a partial synthesis of ß-galactosidase. This explains why, in the

absence of IPTG, there is some elevated ß-galactosidase synthesis. When IPTG

binds to the repressors with increased affinity, it lowers the affinity back to that

of the normal repressor (without IPTG bound). Then, the repressor can rapidly

dissociate from sequences in the chromosome that mimic the operator and find

the true operator. Thus, ß-galactosidase is repressed in the presence of IPTG in

strains with repressors that have greatly increased affinity for operator. In

summary, because of a kinetic phenomenon, we see a reverse induction curve.

• The lacY gene produces a permease that transports lactose into the cell.A cell containing a lacY–mutation cannot transport lactose into the cell, so ß-galactosidase will not be induced. (In wild-type cells, even when the repressor is present, there is still a small amount of transcription. This allows a small amount of baseline permease (and β-gal) expression. It is this low level of permease that allows trace amounts of lactose to enter the cell and initiate induction of the lac operon.)

Associated with the lactose (lac) operon are a series of genetic signals that determine when and how often expression of the lac operon occurs. One of these signals is the promoter (P) where transcription initiation is thought to occur . Another signal is the operator (0), the target or binding site for the lac repressor . Genetic experiments have indicated that these signals are arranged in the following order, I-P-0-Z, where I and Z are the structural genes for the repressor and ,6-galactosidase, respectively, and that the operator is clearly separated from P and Z . The development of in vitro systems for studying repressor-operator interaction by Gilbert and Muller-Hill and Riggs et al. And the existence of specialized transducing phages containing mutations known to define the lacP and/or lacO regions has facilitated the possibility of determining in vitro whether the conclusions of the genetic experiments are in fact correct. The experiments described in this communication present such an analysis. The results confirm the observation that operator constitutive mutations reduce repressor binding to lac DNA in vitro they indicate that promoter point mutations and deletions do not measurably affect the repressor-operator interaction; and they show that deletion of the entire Z gene does not affect the repressor-operator interaction. A surprising resi4lt of these studies is that, although the operator is the primary binding site for the lac repressor, it is not the only one. Indeed there is a secondary lac repressor-binding site located within the a region of the lacZ gene.

(NOTE : In the lac operon, lactose binds to the repressor protein and prevents it from repressing gene transcription, while in the trp operon, tryptophan binds to the repressor protein and enables it to repress gene transcription.)