I am trying to synthesize a protein (with 6 amino acids) in a microcentrifuge tu

Question

I am trying to synthesize a protein (with 6 amino acids) in a microcentrifuge tube (it is actually possible to do this -in vitro synthesis) a) Make a list of ALL components I would have to add to the tube to make this work (4 points) b) Ineed to use mRNA-how do I protect the mRNA in this synthesis from being destroyed? (2 points) When I add the following mRNA strand, the protein that is made is different than what was expected. The mRNA strand that I add is: c) 5' end AUGCCACGCUCUGCGCAU 3' end What I do get is a protein, 5 amino acids in length that begins with Cys (Cysteine) Tell me 1 the amino acid sequence of the protein I am trying to synthesize from the original strand (2 points) 2) the amino acid sequence of the protein I produced (2 points) happened to produce the unexpected protein sequence? what (2 points)

Explanation / Answer

a. In order to synthesize a protein of 6 amino acid, in vitro protein synthesis can be carried out. This is also known as a cell free protein synthesis. Bothe transcription and translation occurs in same tube. Hence, component for both transcription and translation should be added. However, if mRNA is added as template, then only translation components are added to tube. I have included components of both transcription and translation.

For this, the following chemical and reagents are needed.

i) Cell Extract or isolated DNA

ii) Magnesium and other cofactors

iii) Ribosomes

iv) ribonuclotides (rNTPs) for mRNA synthesis

v) tRNAs corresponding to six amino acid

vi) T7 RNA polymerase for transcription of gene of interest. If there is linear DNA, T7 terminator is also required.

vii) aminoacyl-tRNA synthetases

viii) Energy regeneration enzymes and translation factors

ix) RNase inhibitor

x) 6 Amino acids

b) In order to protect RNA, an RNAse inhibitor is added to the mix. The RNase inhibitor will prevent RNases A, B and C from acting on mRNA and total RNA and degrading it. RNase inhibitors bind to RNases by forming tight complexes.

c) 5’ AUG CCA CGC UCU GCG CAU 3’

1) 5’ AUG CCA CGC UCU GCG CAU 3’

Protein expected to be formed starts with AUG, which is the initiator codon coding for methionine.

Protein sequence as per the codon chart is:

Methionine-Proline-Arginine-Serine-Alanine-Histidine

Or

Met-Pro-Arg-Ser-Ala-His

2) The first codon should be UGU or UGC. Hence, The A of the sequence is not considered for protein synthesis.

The mRNA sequence that gives rise to this truncated protein is

5’ UGC CAC GCU CUG CGC AU 3’

Protein sequence is:

Cys-His-Ala-Leu-Arg

The AU present are only 2 nucleotides and hence, will not code for an amino acid.

3) An alternative start site for protein translation was utilized by the ribosomes. The ribosome used UGU as the translation start site. The first adenine was ignored during translation. A cysteine tRNA binds to mRNA instead of the traditional methionine initiator tRNA. Hence, the readings sequence was altered, and the amino acid sequence produced was completely different from the original expected one.

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