Any input would be greatly appreciated! This is for a Molecular Biology class an
ID: 60676 • Letter: A
Question
Any input would be greatly appreciated!
This is for a Molecular Biology class and we just went over Transcription and Translation in great detail.
If a gene is cloned and then expressed in vitro (via a coupled transcription/translation system), the expectation is that a single polypeptide will be observed when analyzed by SDS Page (e.g., at 60 kD in lane 1). Furthermore, if a premature stop codon were engineered 83% of the way down the gene, then protein synthesis would stop prematurely and a single polypeptide would be expected at 50kD (lane 2). In a number of cases, a "gene" was thought to be cloned, but when that "gene" was expressed in vitro in fact two proteins were observed via SDS Page. In this problem you will learn of two different ways that this can happen. In both cases (Operon A and Operon Z), the two proteins are 60 kD and 40 kD MW. Refer to these as A60 and A40 for the proteins from Clone A, which contains Operon A. Refer to these as Z60 and Z40 for the proteins form Clone Z, which contains Operon Z. Operons A and Z are different, and have nothing to do with each other. The results in lane#2 for Clones A and Z were obtained when a single premature stop codon was engineered into the clone. The results in lane#3 for panels A and Z were obtained when a single premature stop codon was engineered into the clone, but the premature stop codon was located at a different position than used for lane #2.Explanation / Answer
1.
The bands in lane 2 of panel A were obtained at 60kD and 30kD. One band is normal as usual, but in the other band, the stop codon has introduced bit near and produced very short protein, which is 10kD less. Thus, produced band at 30kD. In lane 3, the stop codon got introduced exactly at the middle of the gene producing two bands of equal size. (60+40 = 100/2 = 50). Therefore, we could see bands at 50kD.
2.
In lane 2 of panel Z, the stop codon got introduced twice at two different positions, producing 3 bands of equal size. Hence, we could large band (1+1+1) at 30kD. In lane 3, one band is as usual at 40 kD, but the other one is at 50kD. It means, the premature stop codon got introduced 83% of the way down the gene, producing a polypeptide that is 10kD less in size.
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