Questions

1. Given this information and what you’ve learned from your project, explain why it might be important to study antibiotic resistance by looking at protein folding and mis-folding.

Translation occurs within a ribosome. Protein folding often begins at the N-terminus while the rest of the mRNA is still being translated. Since the ribosomes of bacteria are structurally different from those found in eukaryotic cells, antibiotics often target ribosomes which can inhibit protein synthesis of the bacteria, ultimately killing it. If bacteria are becoming resistant to antibiotics, there must be something occurring to stop the inhibition of the ribosome. Antibiotics can also lead to protein mis-folding, which would mean that antibiotic resistance is affecting correct folding (somewhat indirectly). If there are diseases that are caused by protein misfolding, perhaps studying antibiotic resistance would provide insight into how to treat these diseases.

2. Why would the sequences of the NBD₂ determinants (prokaryotic) be similar to the eukaryotic ABCF subfamily?

This suggests that these proteins have similar functions. Since the ABCF subfamily of proteins has been shown to be associated with ribosomes, it is logical that the NBD₂ proteins may also associate with ribosomes (to confer antibiotic resistance) although this has not been currently demonstrated.

3. Even the mechanism of the antibiotic resistance determinants comes down to proteins (or the cessation of their production). Which of the alternative hypotheses expressed in this paper (see text and Figure 1) do you consider the best explanation for this mechanism?

We believe that the ‘cuckoo’ hypothesis is currently the best explanation. It has the most direct evidence to support it. One study using co-localization experiments showed Vga(A) to be associated with the cell membrane. Another study showed a reduction of radiolabelled erythromycin when transformed by the NBD₂ encoding gene msr(A). The ‘ribosome protection’ hypothesis has only indirect evidence supporting it thus far.

1. The authors refer in the introduction to their capacity to “fine-tun[e] the selective pressures in ways not permitted by the cellular environment.” To what selection pressures might they be referring?

There are several pressures that they might be referring to. The first, and most relevant to this article, are unnatural substrates (such as antibiotics). Other selection pressures might be dramatically changed temperatures and a selection that deletes certain translation components. These pressures may not be possible to do in vivo but may give insight into what is occurring in living animals.

2. The discussion focuses on two mutations (one transition and one transversion) that confer resistance to the MLS family of antibiotics (the same family discussed in the other paper). How could these two little mutations change the ribosome sufficiently to confer antibiotic resistance?

These two mutations might seem insignificant but are capable of changing the 3-D structure of the ribosome. In this case, the mutations seem to alter the part of the ribosome to which the MLS family of antibiotics binds to inhibit the function of the ribosome, without altering the function of the ribosome itself. If the antibiotic can’t bind to the ribosome, the ribosome will continue to function, and antibiotic resistance has just occurred.

3. Why might the single-mutant ribosomes be less common in the population than the double-mutant ribosomes?

The authors suggest that there may be subtle differences in activity between the two mutants that cannot be seen in the in vitro assays. This seems to be the most likely explanation. It would be interesting to look the 3-D version of both mutant ribosomes. It is possible that the double-mutant is more conducive to antibiotic resistance or that the single-mutant is less likely to survive for some reason. Other possible explanations are that the double-mutant ribosome amplified better during PCR for some reason, a skewed original population, or that selection occurred during the induction period.