Question

3. Consider a hypothetical strain of E. coli (strain 401) that contains two mutations that affect tryptophan biosynthesis. The first mutation is a single nucleotide substitution that converts the second tandem Trp codon in region 1 of the Trp leader sequence into a stop codon (see slides 2-7 of Lecture 24 on iLearn) trp structural genes trpE trpD trpC trpB trpA PO Trp Leader Met-Lys-Ala-lle-Phe-Val-Leu-Lys-Gly-Trp-Trp-Arg-Thr Ser- Stop Assume that strain 401 also contains a mutation in the gene for the Trp aporepressor that abolishes its ability to bind tryptophan and therefore cannot perform the function of the Trp holorepressor A. Describe the single nucleotide substitution that converts the Trp codon into a stop codorn B. Compare how levels of tryptophan in the growth medium would affect the levels of Trp A-E in strain 401 and in wild type E. coli using the tables below. Indicate whether: (a) stem-loops 2-3 or 3-4 would form, (b) whether transcription would be attenuated and (c) how these events affect expression of Trp A-E. (Reviewing Fig 10.15 of the Slonczweski text and animation 10.2 might be helpful.. Wildtype E. coli SL2-3SL3-4Attenuation? Expression of Trp A-E Hi Trp Low Trp E.coli strain 401 SL2-3SL3-4 Attenuation? Expression of Trp A-E Hi Trp Low Trp C. How would the response to the presence or absence of tryptophan in the growth medium differ if strain 401 is transformed with an F plasmid that expresses a Trp repressor capable of performing holorepressor function (strain 401/FtrpR+)? rp A-E expression 401 wildtype 401/F'trpR+ D. How would the response to the presence or absence of tryptophan in the growth medium differ if strain 401 is transformed with an F plasmid that contains a wildtype Trp leader region but does not contain the genes for Trp A-E downstream from the leader region on the F'" factor E. Why would you not expect to see regulatory circuits such as the trp attenuator in eukaryotic cells?

Answer 1

A. The single nucleotide substitution is substitution of a single base of a codon with a different base or nucleotide. In this case, the codon for the tryptophan amino acid (UGG) is changed due to a change a single nucleotide which may be the 2nd nucleotide. The G may have been substituted by A, which lead to UAG which is a stop codon. Thus, the Trp is changed to stop.

B. Generally, Trp operon is repressible operon, which means that when tryptophan is available the operon is repressed and the biosynthetic pathway is turned off.

Therefore in case of wild type E. coli, when the medium contains high levels of tryptophan, the operon is switched off. (a) There will be no stem loop structure in the regions 2 and 3 as this encodes anti-termination.

(b) The regions 3 and 4 will form a stem loop structure and transcription will be attenuated.

(c) Also, the expression if the Structural genes A-E will be turned off.

when the medium contains low levels of tryptophan, the operon is switched on as the tryptophan (an aporepressor ) is unable to bind to the repressor, threfore the operon is not turned off. (a) There will be a stem loop structure in the regions 2 and 3 as this encodes anti-termination.

(b) The regions 3 and 4 will not form a stem loop structure and there will be no transcription attenuation.

(c) Also, the expression if the Structural genes A-E will be turned on.

In case E. coli strain 401, contains a stop codon in the leader sequence, also it does not contain an aporepressor. So the polymerase will bind to the promoter , but the transcription of the leader sequence (region 1 ) won't be completed. This will lead to the stem loop structure of region 1 and 2 and pause the transcription.

(a) There will be no stem loop structure in the regions 2 and 3 as this encodes anti-termination.

(b) The regions 3 and 4 may form a stem loop structure and transcription will be attenuated.

(c) Also, the expression of the Structural genes A-E will be turned off.

C. In the presence of tryptophan, in case of 401/F' the A-E expression will be turned OFF , in case of strain 401, the A-E expression will be turned OFF ( since the polymerase is unable to move forward and cause transcription of the leader sequence), and in case of wild type, the A-E expression is turned OFF.

In the absence of tryptophan, in case of 401/F' the A-E expression will be turned ON , in case of strain 401, the A-E expression will be turned OFF, and in case of wild type, the A-E expression is turned ON.

D. If the strain 401 has wild type Trp leader sequence , but doesn't have a Trp aporepressor, the operon will be not be turned off and there will be expression of A-E. However, if it contains the wildtype aporepressor, then the operon will be swtiched off.

E. Eukaryotic genes are monocistronic and each has its own promoter, unlike the prokaryotic operons which is assemblage of many genes functioning under one promoter making them polycistronic. Hence, we do not expect to see such regulatory circuits in eukaryotes. In eukaryotes, attenuation doesn't occur. Instead to switch off the expression if a gene, mRNA silencing occurs.