Attenuation


Yanofsky's lab isolated two different mutants with mutations in the trp leader-attenuator region. The mutants are shown below as they would affect the trp leader RNA. Mutation #1 is a base substitution mutant and mutant #2 is a deletion which deletes the indicated sequences. The effects of these mutations on expression of the trpE structural gene are shown in the table below.

StraintrpE Activity
trpR trpL+ (parent)1.0
trpR trpL #10.1
trpR trpL #20.1
trpR trpL #1 pseudorevertants2.5 - 4.0
trpR trpL #2 pseudorevertants2.5 - 4.0

A. How can the effects of mutations #1 and #2 be explained at the molecular level?

B. Yanofsky's lab also isolated "revertants" of mutants #1 and #2. The trpE expression in these mutants is also shown in the table below. Some revertants, in the case of mutant #1, were true revertants in that the wild type base was restored. However, several mutants were "pseudorevertants" in that they still retained the #1 or #2 mutations but also contained a new mutation. Assuming that the pseudorevertants are base substitutions mutations, where are they likely to map in the RNA sequence shown below? How do they affect trp expression at the molecular level?


The trpS gene codes for tryptophanyl tRNA synthetase, the enzyme which aminoacylates tryptophanyl tRNA with tryptophan. trpS does not map near the trp operon. Some trpS mutants have been isolated which show increased expression of the trp operon enzymes. Explain briefly:

i. The nature of the trpS mutation (i.e. how is the mutant different from the wild type enzyme). Remember, a knock-out mutant would be lethal.

ii. The mechanism behind the overexpression of the trp enzymes.


You have isolated 7 deletions that delete various portions of the trp operon regulatory region. A map of the trp regulatory region is shown below. The DNA deleted in each mutant is indicated by the brackets ( ). Each strain is also trpR-. You grow each mutant in media supplemented with excess tryptophan and assay the levels of the trpE protein made by the cells. What would be the effect of each deletion mutation on expression of trpE? You can express your answer in terms relative to a wild type strain grown in excess tryptophan if you wish. Explain each answer briefly in terms of molecular interactions you would expect to occur.


You have isolated a mutant within the following properties: it is a tryptophan auxotroph, and the mutation maps in the trpR gene. Propose a model that explains the mutant phenotype at the molecular level. What type of mutation (not in trpR) could you introduce into this strain to make it trp+ again?


Charles Yanofsky's lab isolated two different types of mutants which each have mutations in the trp leader-attenuator region (trpL). The mutants are shown below as they would affect the trp leader RNA.

Mutation #1 is a base substitution mutant and mutant #2 is a deletion which removes the indicated sequences. The effects of these mutations on expression of the trpE structural gene are shown in the table below.

StraintrpE Activity
trpR- trpL+ (parent)1.0
trpR- trpL #10.1
trpR- trpL #20.1

(a) How can the effects of mutations #1 and #2 be explained at the molecular level?

Yanofsky's lab also isolated revertants of mutants #1 and #2. Some revertants, in the case of mutant #1, were true revertants that restored the wild-type base sequence. However, several mutants were "pseudorevertants" that retained the #1 or #2 mutations but also contained a new mutation. The trpE expression in these mutants is shown in the table below.

StraintrpE Activity
trpR- trpL #1 pseudorevertant2.5 - 4.0
trpR- trpL #2 pseudorevertant2.5 - 4.0

(b) Assuming that the pseudorevertants are base substitutions mutations, where are they likely to map in the trpL RNA sequence and how would they affect trp expression at the molecular level?


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Last modified October 30, 2003