Any input on this would be very welcome but a more simple answer would be better

Question

Any input on this would be very welcome but a more simple answer would be better since I am having a difficult time understanding what this question wants.

You have made an interesting observation concerning overexpression of phage int gene. In lab, you made a plasmid construct that placed the int gene downstream of a strong regulatable promoter and supplied a strong ribosome binding site for the mRNA transcript encoding int. When int expression was turned on from this construct, protein synthesis was inhibited and cells died. However, you showed that cell death was not due to toxicity of the Int protein produced, because the deleterious effects of int overexpression is reversed if cells contain a plasmid expressing an arginyl tRNA gene, which makes a tRNA that decodes the AGA codon.

You also noticed this pattern of usage of arginine codons in the Int protein (shown in table below).

From the information provided, what do the results suggest about the mechanism of overexpression toxicity?

codon Number of codons in int frequancy of usage in E. coli Protiens CGU 2 0.42 CGC 6 0.37 CGA 5 0.05 CGG 1 0.08 AGA 10 0.04 AGG 9 0.03

Explanation / Answer

int gene of Lamda contains high frequency of the rare arginine codons like AGA and AGG, as well as dual rare Arg codons at three positions. Int protein expression depends on the rare AGA tRNA.

int expression was turned on from the above said construct, protein synthesis was inhibited and cells died. However, the deleterious effects of int overexpression is reversed if cells contain a plasmid expressing anarginyl tRNA gene, which makes a tRNA that decodes the AGA codon.

This clearly indicates that reduction of the rare Arg tRNA due to ribosome stalling or delaying at multiple AGA codons on the overexpressed int mRNA underlies all of these observable facts. Dependency of mRNA on rare codon inhibits protein synthesis and cell growth. These results point out that care should be taken in efforts to clone and over express heterologous genes with codon biases.

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