Seven E. coli mutants were isolated. The activity of the...

Seven $E$. coli mutants were isolated. The activity of the enzyme $\beta$ -galactosidase produced by cells containing each mutation alone or in combination with other mutations was measured when the cells were grown in medium with different carbon sources. $$\begin{array}{lcrc} & & & \text { Lactose }+ \\ & \text { Glycerol } & \text { Lactose } & \text { Glucose } \\ \hline \text { Wild type } & 0 & 1000 & 10 \\ \text { Mutant 1 } & 0 & 10 & 10 \\ \text { Mutant 2 } & 0 & 10 & 10 \\ \text { Mutant 3 } & 0 & 0 & 0 \\ \text { Mutant 4 } & 0 & 0 & 0 \\ \text { Mutant 5 } & 1000 & 1000 & 10 \\ \text { Mutant 6 } & 1000 & 1000 & 10 \\ \text { Mutant 7 } & 0 & 1000 & 10 \\ \mathrm{F}^{\prime} \text { lac from mutant } & 0 & 1000 & 10 \\ \text { 1/ mutant 3 } & & & \\ \text { F' lac from mutant } & 0 & 10 & 10 \\ \text { 2/ mutant 3 } & & & \\ \text { Mutants 3 + 7 } & 0 & 1000 & 10 \\ \text { Mutants 4 + 7 } & 0 & 0 & 0 \\ \text { Mutants 5 + 7 } & 0 & 1000 & 10 \\ \text { Mutants 6 + 7 } & 1000 & 1000 & 10 \end{array}$$ Assume that each of the seven mutations is one and only one of the genetic lesions in the following list. Identify the type of alteration each mutation represents. a. superrepressor b. operator deletion c. nonsense (amber) suppressor tRNA gene (assume that the suppressor tRNA is $100 \%$ efficient in suppressing amber mutations) d. defective CRP-cAMP binding site e. nonsense (amber) mutation in the $\beta$ -galactosidase gene f. nonsense (amber) mutation in the repressor gene g. defective crp gene (encoding the CRP protein)

Seven $E$. coli mutants were isolated. The activity of the enzyme $\beta$ -galactosidase produced by cells containing each mutation alone or in combination with other mutations was measured when the cells were grown in medium with different carbon sources.
$$\begin{array}{lcrc}
& & & \text { Lactose }+ \\
& \text { Glycerol } & \text { Lactose } & \text { Glucose } \\
\hline \text { Wild type } & 0 & 1000 & 10 \\
\text { Mutant 1 } & 0 & 10 & 10 \\
\text { Mutant 2 } & 0 & 10 & 10 \\
\text { Mutant 3 } & 0 & 0 & 0 \\
\text { Mutant 4 } & 0 & 0 & 0 \\
\text { Mutant 5 } & 1000 & 1000 & 10 \\
\text { Mutant 6 } & 1000 & 1000 & 10 \\
\text { Mutant 7 } & 0 & 1000 & 10 \\
\mathrm{F}^{\prime} \text { lac from mutant } & 0 & 1000 & 10 \\
\text { 1/ mutant 3 } & & & \\
\text { F' lac from mutant } & 0 & 10 & 10 \\
\text { 2/ mutant 3 } & & & \\
\text { Mutants 3 + 7 } & 0 & 1000 & 10 \\
\text { Mutants 4 + 7 } & 0 & 0 & 0 \\
\text { Mutants 5 + 7 } & 0 & 1000 & 10 \\
\text { Mutants 6 + 7 } & 1000 & 1000 & 10
\end{array}$$
Assume that each of the seven mutations is one and only one of the genetic lesions in the following list. Identify the type of alteration each mutation represents.
a. superrepressor
b. operator deletion
c. nonsense (amber) suppressor tRNA gene (assume that the suppressor tRNA is $100 \%$ efficient in suppressing amber mutations)
d. defective CRP-cAMP binding site
e. nonsense (amber) mutation in the $\beta$ -galactosidase gene
f. nonsense (amber) mutation in the repressor gene
g. defective crp gene (encoding the CRP protein)

Key Concepts

Lac Operon Regulation

The lac operon is a classic model of gene regulation in bacteria, comprising genes involved in lactose metabolism. Its expression is controlled by both negative regulation, through the binding of a repressor to the operator, and positive regulation, via the binding of the CRP-cAMP complex to the promoter region. This dual control allows the cell to integrate signals about the presence of lactose and the availability of glucose.

Negative Regulation and Superrepressor Mutations

Negative regulation occurs when a repressor protein binds to the operator sequence to block transcription. In some mutations, a change in the repressor leads to a superrepressor phenotype, where the protein becomes insensitive to the allosteric effects of the inducer (lactose), thus continuously repressing transcription regardless of environmental signals.

Operator Mutations

Mutations affecting the operator, such as deletion of the operator sequence, eliminate the binding site for the repressor. This disruption results in the loss of repression control and can lead to constitutive expression of the downstream genes, as the repressor is unable to inhibit transcription.

Nonsense Mutations

Nonsense mutations introduce a premature stop codon into the coding sequence, causing the synthesis of truncated, nonfunctional proteins. Such mutations can occur in genes encoding critical proteins like ?-galactosidase or regulatory proteins such as the lac repressor, thereby disrupting normal operon function.

Suppressor tRNA

Suppressor tRNAs are altered transfer RNAs capable of recognizing stop codons introduced by nonsense mutations and inserting an amino acid instead of terminating translation. This can restore the production of full-length, functional proteins, effectively overriding the effect of the nonsense mutation.

CRP-cAMP Mediated Activation

The CRP-cAMP system is essential for the activation of the lac operon, particularly under low-glucose conditions. The cAMP receptor protein (CRP) binds cAMP to form a complex that attaches to the promoter region, thereby enhancing RNA polymerase binding and promoting transcription. Mutations either in the crp gene or in the cAMP binding site can result in reduced or abolished transcriptional activation.

Transcript

00:01 To use lactose, you need lacy permease plus lacz, bata ,gal, and the operand must be expressed.

00:07 Lactose must get into the cell and be broken down.

00:11 Lack y imports lactose, lactase, cleaves lactose into usable sugars.

00:15 If the operon can't be expressed, you won't make either protein.

00:19 And so now you check the mutation.

00:22 Deletion of lack y means no permease can't use lactose, yes.

00:26 You've got the operator constitutive, so the operon is on, the gene still intact, that can use lactose, so no.

00:36 For three, miscence in lack z is going to be a defective beta gal, and so you can't use lactose, so that's good.

00:47 For inversion of entire lack operon, it is going to lead to a promoter plus genes put together, so transcription still works, so it can still use.

00:56 Lactose in principle so no the super repressor can't be induced so loprines often can't use lactose so yes and invert likewise zya but not lack lp and o okay so that's promoter points away from gene so no lack zy a transcription so it can't use lactose so prevent lactose use would be one 3, 5, and 6.

01:34 We're told mutant 1 is a deletion of lack y.

01:38 So mutant 1 already has a working lack z.

01:45 So the missing piece is lack y.

01:48 And the complement mutant 1 would be 2.

01:55 Because lack y is present in express.

01:57 That's complements 3.

02:00 Lack y is fine.

02:01 So complements mutant one provides lack z and four still expresses lack y...