Question

b) (2 points) Describe the experiment and the results that allow her to conclude that the five mutants result from mutations in five different genes. 097 m iii) Cysteine, cystathionine and homocysteine are precursors to methionine. She grew all five mutant lines on minimal medium with either cysteine, cystathionine, homocysteine or methionine, with the following results (+ indicates growth of the mutant line, - indicates no growth): + + + + + + Cysteine cystathionine homocysteine methionine met-1 met-2 met-3 met-4 (+ ) met-5 c) (5 points) Based on this information, draw a pathway for the biosynthesis of methionine, indicating the position of all chemical precursors, and the position of all genes (MET-1, MET-2 MET-3, MET-4, MET-5) identified by mutation. + + + + +

Answer 1

iii) We are given that methionine biosynthesis involves precursors like cysteine, cystathionine, and homocysteine. The pathway involves many other precursors and each step involves conversion by a specific enzyme encoded by different genes. In the experiment performed, mutants for genes met-1, met-2, met-3, met-4, and met-5 were developed. These are loss of function mutations, i.e. mutation in these genes leads to failure of the methionine biosynthesis pathway and thus the growth of the bacteria. However, these genes produce products that are involved in specific steps in the pathway. If a compound that is downstream in the pathway is supplemented, the growth will be restored back.

1. The easiest way to decode this mystery pathway is to start with the mutant that can be reverted by most of the substrates. From the table, we can see this is met-4. met-4 mutant when treated with either cysteine, cystathionine, homocysteine or methionine can restore growth. Thus, these products will be produced downstream to the step which utilizes the gene product met-4.

2. Now, the met-3 mutant can be made to grow with the substrates cystathionine, homocysteine or methionine. However, it fails to grow in the presence of cysteine. Thus, met-3 is involved in a step downstream to cysteine but upstream to cystathionine, homocysteine, and methionine. This also tells that cysteine is produced upstream in the pathway compared to the aforementioned precursors.

3. met-1 and met-5 mutants are reverted by the addition of homocysteine and methionine, but not by cysteine and cystathionine. Thus, these two enzymes are involved downstream of cysteine and cystathionine and upstream of homocysteine and methionine. Also, this implies that cysteine and cystathionine are upstream to methionine and homocysteine. Combining this result with the conclusion of 2, we have, cysteine is upstream to cystathionine which is upstream to methionine and homocysteine.

4. met-2 mutants exhibit growth only upon supplementation with methionine. Hence this gene product is involved downstream of cysteine, cystathionine and homocysteine but upstream to methionine. This along with previous results shows that homocysteine is upstream to methionine.

Comparing everything, we have the following pathway at our hands.

met-4 Cysteine met-3 Cystathionine met-1 met-5 Homocysteine met2 met-2 Methionine