The addition of a " \(5^{\prime}-\operatorname{cap}^{\prime \prime}\) and a \(^{\prime \prime} 3^{\prime}\) poly-A" tail to many nuclear RNA species occurs prior to export of mature mRNAs to the cytoplasm. Such modifications appear to influence mRNA stability. Many assays for gene regulation involve the use of reporter genes such as luciferase from the North American firefly (Photinus pyralis). When the luciferase gene is transcribed and translated, the protein product can be easily assayed in a test tube. In the presence of luciferin, ATP, and oxygen, the luciferase enzyme produces light that can be easily quantified. Assuming that luciferase mRNA can be obtained and differentially modified (5'-capped, poly-A tailed) in a test tube, suggest an assay system that would allow you to determine the influence of \(5^{\prime}-\) capping and poly-A tail addition on mRNA stability.

Step by step solution

01

Isolation of luciferase mRNA

Extract luciferase mRNA from the North American firefly (Photinus pyralis) using standard molecular biology techniques, such as RNA isolation via extraction followed by purification through an RNA-binding column or gel electrophoresis. It's crucial to obtain pure and intact mRNA for subsequent experiments.

02

mRNA Modification

Divide the isolated luciferase mRNA into three groups. The first group will remain unmodified, the second group will have a 5'-cap modification added, and the third group will have a poly-A tail added using appropriate molecular biology tools and enzymes. Ensure that the modifications are correctly added using techniques like RT-PCR or sequencing.

03

In vitro Translation

Generate luciferase protein from each group of mRNA using an in vitro translation system, which includes all essential elements required for protein synthesis, such as ribosomes, tRNAs, and accessory factors. Translation reactions should be performed in triplicate to ensure reproducibility of the results.

04

Incubation and Degradation

Incubate the translated luciferase proteins along with ribonucleases or other molecules that degrade mRNA. Incubate the samples at specific time points (e.g., 0, 2, 4, and 6 hours) to monitor the mRNA stability over time.

05

Measuring Luciferase Activity

After each time point, isolate the remaining mRNA from the protein matrices and use this mRNA in a new in vitro translation system containing luciferin, ATP, and oxygen. Then, measure the light produced by the luciferase enzyme at each time point using a luminometer. This will allow us to determine the amount of translated protein produced from the remaining intact mRNA in each sample group.

06

Analyzing the Results

Comparing the amount of light (i.e., luciferase activity) produced at various time points in each group would allow determining which mRNA type was more stable. If the 5'-capping and poly-A tail addition on mRNA increase mRNA stability, those groups will tend to have a higher luciferase activity over time compared to the unmodified mRNA group. By following these steps, it would be possible to determine the influence of 5'-capping and poly-A tail addition on mRNA stability using the North American firefly (Photinus pyralis) luciferase as a reporter gene.

Unlock Step-by-Step Solutions & Ace Your Exams!

• Full Textbook Solutions

  Get detailed explanations and key concepts

• Unlimited Al creation

  Al flashcards, explanations, exams and more...

• Ads-free access

  To over 500 millions flashcards

• Money-back guarantee

  We refund you if you fail your exam.

Start your free trial

Over 30 million students worldwide already upgrade their learning with Vaia!