Page 150 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

Problems 309

mutant protein can be purified and its amino acid 12. A particular protein has the amino acid sequence
sequence determined. The amino acid sequence of the
mutant protein is exactly the same as the amino acid N . . . Ala­Pro­His­Trp­Arg­Lys­Gly­Val­Thr . . . C
sequence of the wild­type protein from the N termi­ within its primary structure. A geneticist studying
nus of the protein to the glycine in the preceding se­ mutations affecting this protein discovered that sev­
quence. Starting with this glycine, the sequence of eral of the mutants produced shortened protein mole­
amino acids is changed to the following: cules that terminated within this region. In one of
them, the His became the terminal amino acid.
N . . . Gly His Gln Gly Lys . . . C
a. What DNA single­base change(s) would cause the
Using the amino acid sequences, one can determine protein to terminate at the His residue?
the sequence of 14 nucleotides from the wild­type
gene encoding this protein. What is this sequence? b. What other potential sites do you see in the DNA
9. The results shown in Fig. 8.5 may have struck you sequence encoding this protein where mutation of
a single base pair would cause premature termina­
as incongruous because many synthetic RNAs that tion of translation?
lacked AUG start codons (such as poly­U) were none­
theless translated into polypeptides in vitro. The reason 13. How many possible open reading frames (frames
this experiment was possible is that Marshall Nierenberg without stop codons) exist that extend through the fol­
2+
found that a high concentration of Mg ions in the lowing sequence?
test tube, much higher than that found in cells, allows 5′... CTTACAGTTTATTGATACGGAGAAGG...3′
ribosomes to initiate translation at any position on an 3′... GAATGTCAAATAACTATGCCTCTTCC...5′
RNA molecule. Predict the outcomes of in vitro trans­ 14. a. In Fig. 8.3, the physical map (the number of base
lation with each of the following synthetic mRNAs at pairs) is not exactly equivalent to the genetic map
2+
both high and low Mg concentrations: (in map units). Explain this apparent discrepancy.
a. poly­UG (UGUGUG . . .) b. In Fig. 8.3, which region shows the highest fre­
b. poly­CAUG (CAUGCAUGCAUG . . .) quency of recombination, and which the lowest?
−
c. poly­GUAU (GUAUGUAUGUAU . . .) 15. Charles Yanofsky isolated many different trpA
10. Identify all the amino acid­specifying codons in the mutants (Fig. 8.3).
genetic code where a point mutation (a single base a. Explain how he could identify Trp auxotrophs of
−
change) could generate a nonsense codon. E. coli using replica plating (recall Fig. 7.6).
11. Before the technology existed to synthesize RNA b. Assuming that the role of TrpA enzyme in the
molecules of defined sequence like those in Fig. 8.5, tryptophan biosynthesis pathway was known, explain
similar experiments were performed with synthetic how Yanofsky could have identified trpA mutants
−
−
mRNAs of undefined sequence. For example, RNAs among his Trp auxotrophs. (Hint: Recall Beadle
consisting only of Us and Gs could be synthesized in and Tatum’s one gene, one enzyme experiments in
vitro, but they would have random sequences. Suppose Chapter 7.)
a pool of random­sequence RNAs was synthesized in 16. The sequence of a segment of mRNA, beginning with
a reaction mixture containing three times as much UTP the initiation codon, is given here, along with the cor­
as GTP, and that the resulting RNAs were translated responding sequences from several mutant strains.
in vitro. Normal AUGACACAUCGAGGGGUGGUAAACCCUAAG...
a. How many different codons exist in the RNAs? Mutant 1 AUGACACAUCCAGGGGUGGUAAACCCUAAG...
b. How many different amino acids would you find in Mutant 2 AUGACACAUCGAGGGUGGUAAACCCUAAG...
the polypeptides synthesized?
c. Why are your answers to (a) and (b) not the same? Mutant 3 AUGACGCAUCGAGGGGUGGUAAACCCUAAG...
d. How often would you expect to find each of the Mutant 4 AUGACACAUCGAGGGGUUGGUAAACCCUAAG...
codons in (a)? Mutant 5 AUGACACAUUGAGGGGUGGUAAACCCUAAG...
e. In what proportions would you expect to find each Mutant 6 AUGACAUUUACCACCCCUCGAUGCCCUAAG...
of the amino acids in the polypeptides?
f. If you did this experiment—that is, synthesized a. Indicate the type of mutation present in each, and
random­sequence RNAs containing a 3:1 ratio of translate the mutated portion of the sequence into
U:G, and quantified the amount of each amino acid an amino acid sequence in each case.
in the polypeptides produced—prior to knowledge b. Which of the mutations could be reverted by
of the genetic code table, what would the results treatment with EMS (ethylmethane sulfonate;
have told you? see Fig. 7.14)? With proflavin?