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Why kanamycin causes more Phe than Leu to be incorporated into proteins in cell free system?

Why kanamycin causes more Phe than Leu to be incorporated into proteins in cell free system? - Protocols and Methods Forum

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Old 12-14-2005, 04:57 PM
Simon
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Default Why kanamycin causes more Phe than Leu to be incorporated into proteins in cell free system?



Hi, Everyone!

I have a cell free system S30 extract. There is some endogenous mRNA in
the system. This mRNA is supposed to be random. I add kanamycin. It
causes misreading of mRNA by ribosome. In two separate experiments, I
add labeled Phe and labeled Leu. Only two codons code for Phe but 6
codons code for Leu. Therefore, as a result of misreading of endogenous
mRNA caused by kanamycin, Leu should be more often incorporated than
Phe. But the result shows that more Phe is incorporated into proteins.

Why?

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Old 12-14-2005, 06:52 PM
Peter Ellis
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Default Why kanamycin causes more Phe than Leu to be incorporated into proteins in cell free system?

[Only registered users see links. ] wrote:

Random in what way? Do you mean the sequence itself is random?


Your logic is flawed. It depends on the mechanism of the misreading,
and on your starting population. Kanamycin disrupts wobble pairing.

In the case of codons beginning CU, these all code for leucine, so this
is irrelevant. All these four codons will generate leucine whether or
not kanamycin is present.

In the case of codons beginning UU, two (UUA and UUG) code for leu and
two (UUU and UUC) code for phenylalanine. Since there are equal
numbers of each, wobble mispairing will lead to a symmetrical degree of
misincorporation, which shouldn't affect the ratio of one to the other.


Either:

1) Your mRNA is not truly random
2) Your translation system does not truly reproduce the codons present
in the mRNA
3) There is a systematic bias in which wobble mispairings are promoted
by kanamycin

Of these, I think the most likely is option (2), with a dash of option
(1). It's possibly that a long polynucleotide tract such as
UUUUUUUUUUUUU may cause problems with the translation system. This
means that phenylalanine will be on balance underrepresented. When you
add kanamycin, this underrepresentation would be shared equally between
phenylalanine and leucine, leading to an apparent increase in
phenylalanine incorporation.

Depending on your mRNA source, it may also be that polynucleotide
tracts are underrepresented in the starting mRNA, with the same
consequences.

To check whether this explanation is right, you could also look for
shifts in the lysine/asparagine ratio (AAA/AAG & AAT/AAC). All CCN
codons are proline, and all GGN codons are glycine, so we won't derive
any information from looking at these.

What I'm not clear on is why you're looking for an effect in the first
place, since if the biology all works correctly there shouldn't be one.

Peter
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cell , free , incorporated , kanamycin , leu , phe , proteins , system


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