How would transcription of the E. coli trp operon be affected by the following m
ID: 90667 • Letter: H
Question
How would transcription of the E. coli trp operon be affected by the following manipulations of the leader region of the trp mRNA? (a) Increasing the distance (number of bases) between the leader peptide gene and sequence 2 (b) Increasing the distance between sequences 2 and 3 (c) Removing sequence 4 (d) Changing the two Trp codons in the leader peptide gene to His codons (e) Eliminating the ribosome-binding site for the gene that encodes the leader peptide (f) Changing several nucleotides in sequence 3 so that it can base-pair with sequence 4 but not with sequence 2Explanation / Answer
The trp operon is subject to regulation both by repression and by attenuation. Attenuation depends on the tight coupling between transcription and translation in bacteria. When the trptophan is high in the medium, translation of the trp leader is completed and the ribosome blocks sequence 2 which allows the transcribed sequences 3 and 4 to form the stemloop attenuator structure. As a reult formation of 3:4 loop terminate the transcription of structural genes (EDCBA). But when the trptophan is scarce, translation of the trp leader is stalled at two Trp codons at sequence 1. In this position, the ribosome does not cover sequence 2. As a result sequence 2 can now basepairs with sequence 3 in an alternative secondary structure. Formation of the 2:3 stemloop inhibits formation of the 3:4 attenuator loop, and transcription proceeds on through the trp EDCBA genes. Thus when the bacteria has a low trptophan, the biosynthetic genes are expressed and more Trp is synthesized.
a) If distance increases between leader peptide gene and sequence 2, it will decrease attenuation under conditions of high trptophan. In this situation, the ribosome, after completing translation of the leader, will not cover sequence 2. Hence the 2:3 stemloop can form and it prevents to form 3:4 loop and thereby losing the normal attenuation with a greater distance between sequences 1 and 2.
b) If the distance between sequences 2 and 3 increase, 2:3 loop does not form, rather 3:4 loop (attenuator structure) will form . Thus when the trptophan was low, even though the ribosome has stalled, the 2:3 loop would not form, allowing the 3:4 attenuator structure to form with the result of a decrease in trp operon expression (due to attenuation) even in low trptophan.
c) As sequence 4 is required to form the 3:4 attenuator stemloop in high tryptophan condition, in its absence no attenuation would be observed.
d) If tryptophan is low in the medium, the ribosome will tend to pause at the two trp codons at sequence 1 which leads to form 2:3 loop. As a result transcription of leader sequence cannot complete. But if there two trp codons change into his codons, ribosome will never pause at sequence 1 even in low tryptophan. As a result 3:4 attenuator structures will form and transcription of structural gene become halted.
e) If ribosome binding site eliminated from leader structure, then 3:4 attenuator structure will never form even in high tryptophan condition. As a result transcription of structural genes (EDCBA) will never halt even in high tryptophan level.
f) Changing several nucleotides in sequence 3 so that it can base-pair only with sequence 4 but not with sequence affects only in low tryptophan condition. Because when tryptophan is high sequence 3 normally binds with sequence 4 but in low tryptophan condition sequence 3 binds with sequence 2 to form 2:3 loop. So, in low tryptophan condition if several nucleotides in sequence 3 become change it will never form 2:3 loop. As a result 3:4 attenuator structure will form and it inhibits trancrption of structural gene.
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