Original subtitles

The overall goal of this TOE

printing analysis is to observe the

translation initiation complex formation

on mammalian mrnas. This

method can help answer key questions

in the field of mRNA translation

such as Iris mediated translation

initiation. The main advantage of this

technique is that one can perform

detailed analysis of translation

initiation complex formation on a

specific mRNA.

Assemble the transcription reactions to

transcribe RNA from the PCR

amplified DNA templates encoding the

Ziap iris RNA or its

mutants. Next, add

2 units of Rnase free Dnas

to remove the DNA template to

each 20 microliter reaction of RNA.

Incubate at 37°C for 30

minutes. Then

dilute the DNA's treated RNA with

nuclease free water to a final

volume of 110 microliters.

Next, add 110 microliters of

acid phenol to begin RNA

purification. Vortex the

samples for five seconds.

And centrifuge at 20,000 * g for

three minutes at room temperature. It's

critical to dilute the RNA or cDNA prior

to phenol extraction. If the volume is

too low, it's going to be difficult to

physically recover the aqueous phase and

the final yield will be low.

Then carefully transfer 100 microliters

of the RNA containing aqueous phase

to a new microcentrifuge tube.

Add 10 microliters of three molar

sodium acetate, pH 5.5 and mix

the samples. Then

add three volumes of 100% ethanol

to precipitate RNA. And

vortex the samples for five seconds.

Incubate overnight at

-20°C.

The next day, centrifuged the samples

at 20,000 * g for 30

minutes at 4°C and discard the

supernatant. Then

wash the pellet with 500 microliters

of ice cold 70% ethanol and

repeat the centrifugation.

Carefully aspirate as much supernatant as

possible without dislodging the pellet.

Then air dry the pellet for five to 10

minutes. Resuspend the

pellet in nuclease free water to

a final concentration of 0.5 micrograms

per microliter.

To begin the toe printing assay,

add 15 microliters of non

nuclease-treated rabbit reticulocyte

lysate to each 1.5 milliliter

microcentrifuge tube, which represent the

wild type Ziap iris RNA and

its mutants. Add

Rnase inhibitor.

And GMP PNP to each tube.

Incubate at 30°C for 5

minutes. After this, add 1

microliter of the respective RNA to each

of the tubes. And incubate

at 30°C for an additional 5

minutes. During the

incubation period, add DTT and spermidine

to the toe printing buffer.

Then add 22 microliters of toe printing

buffer to each of the tubes.

And incubate at 30°C for three

minutes. Next, add

0.5 microliters of IR dye

labeled toe printing primer.

And incubate the reaction on ice for 10

minutes. Subsequently add

2 microliters of DNTPS.

Two microliters of magnesium acetate.

And one microliter of avian

myeloblastosis virus reversed

transcriptase. Bring the final

volume to 50 microliters with toe

printing buffer. Then allow primer

extension to occur for 45

minutes at 30°C. It's also

critical to add magnesium acetate to the

toe printing reaction, without which the

reverse transcriptase won't function

optimally. After this, add 200

microliters of nuclease free water.

Add 250 microliters of 25

to 24 to 1 phenol,

chloroform, isoamyl alcohol pH 8.0

to begin the purification of

cDNA. After vortexing

the samples for five seconds.

Centrifuge the samples at 20,000 * g

at room temperature for three minutes.

Next, carefully transfer the aqueous

phase containing the DNA to a

new microcentrifuge tube.

Then add three volumes of 100%

ethanol to precipitate the DNA and

vortex for five seconds.

Incubate overnight at

20°C.

The next day. Centrifuge the

tubes at 20,000 * g for

30 minutes at 4°C.

And discard the supernatant.

Then wash the DNA pellet

with 500 microliters of ice

cold 70% ethanol. And

centrifuge at 20,000 * g for

15 minutes at 4°C.

Aspirate as much supernatant as possible,

taking care not to dislodge the pellet.

Then air dry the pellet for five to

10 minutes.

Dissolve the DNA pellet in six

microliters of nuclease free water.

Then add three microliters of formamide

loading dye.

User Sequencing kit to prepare a

dideoxynucleotide sequencing ladder with

the toe printing primer and the

DNA construct encoding the Ziap iris

RNA. Then mix 6

microliters of each sequencing reaction

with three microliters of the formamide

loading die.

After cleaning and assembling the plates.

Pour the gel.

And insert the comb. Following the

polymerization of the gel, assemble the

sequencing apparatus and fill the

reservoirs with 1X TBE.

Then pre run at 1500 volts

for 15 minutes until a temperature

of 55°C is achieved.

Heat all of the samples with

formamide dye to 85°C for 5

minutes. Then load one

microliter of each sample on a

6% polyacrylamide sequencing gel.

And run the gel at 1500 volts for

8 hours. Finally, read the bands

in real time with a fluorescent based

imager.

This method shows that confirmation

of RNA is critical for

translation initiation. Complex

formation. The reverse transcription of

Ziap mRNA ribosome complex yields

typical TOE prints plus 17

to plus 19 nucleotides downstream

of Aug. The five

prime PPT mutant shows impaired

toeprint formation. However, when

transcribed with the five prime cap

initiation, complex formation occurs.

Similarly, the three prime PPT mutant

also shows impaired toe print formation.

However, transcription with the five

prime cap leads to initiation complex

formation. The double PPT

mutant harboring both the five prime PPT

and three prime PPT mutations is able

to form the initiation complex without

the five-prime cap. Toe print

formation is strongly impaired for the

start codon mutant in the absence

of RRL and GMP PNP and

a polyatail. The cDNA

products are compared with a

dideoxynucleotide sequencing ladder.

Human beta globin or HBB mRNA

shows no toeprint in the absence

of the five prime cap. However,

the initiation complex is formed on

the capped five-prime UTR of HBB,

yielding TOE prints plus 16 to

plus 18 nucleotides downstream of Aug.

Once mastered, this technique can be done

in three days, including preparation of

in vitro transcription templates and RNA

if performed properly. Following this

procedure, other methods like structural

probing can be performed in order to

answer additional questions like the

secondary structure of the wild type or

mutant variants of the iris. After

its development, this technique has paved

the way for researchers in the field of

mRNA translation to explore Iris-mediated

translation in vitro. After watching

this video, you should have a good

understanding of how to perform toe

printing of mammalian mRNA and to analyze

the formation of translation initiation

complexes. Don't forget that acid,

phenol, phenol, chloroform, isoamyl

alcohol, and acrylamide can be extremely

hazardous. Appropriate personal

protective equipment should be worn while

using these reagents.