V. Lab | 80. In vitro transcription and translation

Question 1

I. In vitro transcription
1. What are the features of In vitro Transcription?

Answer 1

(mRNA maturation in eukaryotes: capping, splicing and polyadenylation)
- No RNA processing factors in the in vitro transcription assay are
present
- No cap: the methyl-guanosine cap can be replaced by a GAA
triplet and a tobacco mosaic virus enhancer (omega) sequence
after the GAA triplet in the mRNA
- No Poly-A: a long 3’ untranslated region situated downstream of
the stop codon can amend mRNA stability a great deal
- No splicing: cDNA (copy DNA) of gene of interest is cloned into the
vector

Question 2

I. In vitro transcription
2. What are the events in mRNA maturation in eukaryotes?

Answer 2

capping, splicing and polyadenylation

Question 3

I. In vitro transcription
3. Are there any processing factors in the in vitro transcription?

Answer 3

No RNA processing factors in the in vitro transcription assay are
present

Question 4

I. In vitro transcription
4. In in vitro transcription, there is no cap
=> What can be a substituted solution?

Answer 4

No cap: the methyl-guanosine cap can be replaced by a GAA triplet and a tobacco mosaic virus enhancer (omega) sequence

(mRNA maturation in eukaryotes: capping, splicing and
polyadenylation)

Question 5

I. In vitro transcription
5. In in vitro transcription, there is no Poly-A
=> What can be a substituted solution?

Answer 5

No Poly-A: a long 3’ untranslated region situated downstream of the stop codon can amend mRNA stability a great deal

(mRNA maturation in eukaryotes: capping, splicing and
polyadenylation)

Question 6

I. In vitro transcription
6. In in vitro transcription, there is no splicing-A
=> What can be a substituted solution?

Answer 6

No splicing: cDNA (copy DNA) of gene of interest is cloned into the vector

(mRNA maturation in eukaryotes: capping, splicing and
polyadenylation)

Question 7

II. In vitro translation
1. What are the features of in vitro translation?

Answer 7

Cell-free protein synthesis:
- Protein synthesized in test tubes without living cells
- Translation: ribosome, mRNA, tRNA-aminoacyl, amino acids
=> GFP = green fluorescent protein (used in lab as a marker)

Question 8

II. In vitro translation
2. What does translation reaction include?

Answer 8

Wheat germ extract comprises ribosomes, RNAs, translational factors, aminoacyl-tRNA synthetases and energy sources
- Substrate: amino acid substrate
- Template for translation: mRNA (synthesized in vitro)
- Bilayer: one tube contains everything + one tube contains no RNA polymerase (no mRNA synthesized)