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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Abstract

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

Introduction

RNA binding protein (RBP)-based post-transcriptional regulation, specifically characterization of the interaction between RBPs and RNA, has been studied extensively in recent decades. There are multiple examples of translational down-regulation in bacteria originating from RBPs inhibiting, or directly competing with, ribosome binding1,2,3. In the field of synthetic biology, RBP-RNA interactions are emerging as a significant tool for the design of transcription-based genetic circuits4,5. Therefore, there is an increase....

Protocol

1. System Preparation

  1. Design of binding-site plasmids
    1. Design the binding site cassette as depicted in Figure 1. Each minigene contains the following parts (5' to 3'): Eagl restriction site, ∼40 bases of the 5' end of the kanamycin (Kan) resistance gene, pLac-Ara promoter, ribosome binding site (RBS), AUG of the mCherry gene, a spacer (δ), an RBP binding site, 80 bases of the 5' end of the mCherry gene, and an ApaLI restrict.......

Representative Results

The presented method utilizes the competition between an RBP and the ribosome for binding to the mRNA molecule (Figure 1). This competition is reflected by decreasing mCherry levels as a function of increased production of RBP-mCerulean, due to increasing concentrations of inducer. In the case of increasing mCerulean fluorescence, with no significant changes in mCherry, a lack of RBP binding is deduced. Representative results for both a positive and a negativ.......

Discussion

The method described in this article facilitates quantitative in vivo measurement of RBP-RNA binding affinity in E. coli cells. The protocol is relatively easy and can be conducted without the use of sophisticated machinery, and data analysis is straightforward. Moreover, the results are produced immediately, without the relatively long wait-time associated with next generation sequencing (NGS) results.

One limitation to this method is that it works only in bacterial cells. However, a.......

Acknowledgements

This project received funding from the I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation (Grant No. 152/11), Marie Curie Reintegration Grant No. PCIG11-GA- 2012-321675, and from the European Union's Horizon 2020 Research and Innovation Program under grant agreement no. 664918 - MRG-Grammar.

....

Materials

NameCompanyCatalog NumberComments
Ampicillin sodium saltSIGMAA9518
Magnesium sulfate (MgSO4)ALFA AESAR33337
48 platesAxygenP-5ML-48-C-S
8- lane platesAxygenRESMW8I
96-well platesAxygenP-DW-20-C
96-well plates for plate readerPerkin Elmer6005029
ApaLINEBR0507
Binding site sequencesGen9 Inc. and Twist Biosciencesee Table 1
E. coli TOP10 cellsInvitrogenC404006
Eagl-HFNEBR3505
glycerolBIO LAB071205
incubatorTECANliconic incubator
Kanamycin solfateSIGMAK4000
KpnI- HFNEBR0142
ligaseNEBB0202S
liquid-handling robotic systemTECANEVO 100, MCA 96-channel
Matlab analysis softwareMathworks
multi- pipette 8 lanesAxygenBR703710
N-butanoyl-L-homoserine lactone (C4-HSL)caymanK40982552 019
PBS bufferBiological Industries020235A
platereaderTECANInfinite F200 PRO
Q5 HotStart PolymeraseNEBM0493
RBP seqeuncesAddgene27121 & 40650see Table 2
SODIUM CHLORIDE (NaCL)BIO LAB190305
SV Gel and PCR Clean-Up SystemPromegaA9281
TryptoneBD211705

References

  1. Cerretti, D. P., Mattheakis, L. C., Kearney, K. R., Vu, L., Nomura, M. Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein. Journal of Molecular Biology. 204 (2), 309-329 (1988).
  2. Babitzke, P., Baker, C. S., Romeo, T.

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