The bacterial protein called flagellin (molecular weight 40,000 g/mol) has been

Question

The bacterial protein called flagellin (molecular weight 40,000 g/mol) has been used to calculate the accuracy of protein synthesis. Flagellin offers two advantages, it can be easily obtained as a pure fraction and it contains no cysteine, thereby allowing a sensitive measure of the mis-incorporation of cysteine into the protein.

To radioactively label cysteine, bacteria were grown in the presence of 35SO42-. The specific activity of the radioactive sulfate is 5.0 x 103 cpm/pmole, in other words, one picomole (pmole) of radioactive sulfate produces 5 x 103 counts per minute.

1) After radioactive labeling, 8 ?g of flagellin were purified and were found to contain 300 cpm of 35S radioactivity. Assume that the mass of flagellin doubles during the labeling period and that the specific activity of cysteine in flagellin is equal to the specific activity of 35SO42- used to label the bacteria.
a) Calculate how many pmoles (picomoles) of flagellin have been made during the labeling period.

b) Calculate how many pmoles of cysteines were incorporated in the flagellin synthesized during the labeling period.

2) Of the flagellin molecules that were synthesized during the labeling period, what fraction of flagellin contains cysteine?

3) In flagellin, cysteine is mis-incorporated at the arginine codons CGU and CGC. In term of anticodon-codon interaction, what mistake is made during mis-incorporation of cysteine for arginine?

4) There are 18 arginines in flagellin and all arginine codons (6 total) are equally represented.
a) Calculate the average number of sensitive arginine codons that are contained in a molecule of flagellin.

b) What is the frequency of misreading of each sensitive (CGU and CGC) arginine codon?

5) Assuming that the error frequency per codon calculated above applies to all amino acid codons equally, estimate the percentage of molecules that is correctly synthesized for proteins 100, 1000 and 10000 amino acids in length. (The probability of not making a mistake is one minus the probability of making a mistake).

Explanation / Answer

1)

a) Since, mass of flagellin doubles during the labelling period; therefore 4g of flagellin were produced.

no of moles = mass of flagellin/mol wt of flagellin = 4/40,000 = 10-4 moles = 10-4 * 1012 = 108 pmoles

b) pmoles of Cysteine incorporated = radioactivity of flagellin / specific radioactivity of Cysteine

300 / 5x103 = 0.6x10-1 = 6 pmoles

2)

fraction of flagellin containing cysteine = 6 pmoles / 108pmoles = 0.000006%

3)

codon for cysteine are UGU and UGC. UGC is similar to arginine codon CGC with difference in the first base which is Thymine for cysteine and Cytosine for arginine. Similarly, UGU is for CGU. Error probably occurs during reading these codons i.e. CGC and CGU.

4)

a) The arginine codons are CGU, CGC, CGA, CGG, AGA, AGG. Out of these, two codons are sensitive to misincorporation, CGU and CGC. There are 18 arginine residues in 1 flagellin molecule; all codons being equally represented, i.e. one codon appears 3 times. Therefore, no of sensitive codons for one flagelin molecule is 6.

b) total no of arginine produced(without error) = 18 * no of flagellin molecules = 18 * 10-4 * 6 * 1023 = 1021

total no of cysteine produced = 6x10-12 * 6x1023 = 36x1011

error frequency = 36x1011 / 1021 = 36x10-10 * 100 = 3.6x10-7 %

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