Question

1. OK. So, let's talk about Candyp, a delicious eukaryotic protein that tastes like a Snickers bar. a) You painstakingly derived the (mature) transcribed sequence for Candy, which is listed below. What would be the mRNA sequence that corresponds with this segment? coding 5 TA A TG C AGT AAGC 3' template 3 ATT A C GT CAT I C G 5' b) OK, how you need to get E. coli to make a whole bunch of that protein. What do you need to do to this DNA sequence before inserting it into a plasmid for transformation, to make sure that the E. coli will be able to transcribe this protein? Briefly explain why. c) When the cell goes to translate this mRNA, what would be the amino acid sequence?

Answer 1

1. Given the coding and the template DNA sequence:

coding DNA: 5'- TAATGCAGTAAGC -3'
template DNA: 3'- ATTACGTCATTCG -5'

The mRNA transcription would proceed in the 5' > 3' direction along the template DNA, adding bases that are complementary to the template. Also, mRNA uses Uracil in place of Thymine bases.

The mRNA sequence would therefore be the same as the coding sequence, except for the Thymine bases.

mRNA sequence: 5'- UAAUGCAGUAAGC - 3'

2.  In order to introduce the sequence into the plasmid so that the bacteria is able to transcribe the protein, we need to make sure the entire ORF (Open Reading Frame) is included in the sequence and that the restriction enzyme used for cloning does not cut into the ORF region so it can be inserted into the plasmid as a whole.

3. The amino acid sequence can be determined using the codon table after finding the ORF region in the mRNA, which usually starts with the code AUG for the amino acid Methionine, and ends with a Stop codon which can be UAA , UGA or UAG.

When we look at the mRNA sequence based on the original sequence given above, we can locate the start and the stop codon, marked in bold :

5' - UAAUGCAGUAAGC -3'

The triplet code from AUG can be written for the sequence as:

5' - UA AUG CAG UAA GC -3'

We can determine the amino acid sequence by referring to the codon table, for example, if we take the triplet code AUG, then we locate the codon by locating the first letter A on the left column, U in the central row and G in the right column to find the corresponding amino acid to the triplet code AUG to be methionine.

The amino acid synthesis starts with the N-terminal sequence and ends with the C- terminal.

From the codon table , we find that the triplet code AUG codes for Methionine and the next  CAG codes for glutamine. The amino acid sequence here would be :

Met - Gln - Stop