A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Summary

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Abstract

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Introduction

Gene regulation plays an important role in biology. Genes can be regulated at the level of transcription, pre-mRNA splicing, 3' end formation, RNA export, translation, mRNA localization, decay, post-translational modification/stability, etc. Both DNA- and RNA-binding proteins play key roles in gene regulation. While molecular genetic analyses have identified numerous regulatory proteins, only a small subset of them have been characterized fully for their cellular functions or binding sites in vivo. Phylogenetic sequence analysis and mutagenesis offer complementary approaches to characterize DNA- or RNA-protein interactions.

Protocol

NOTE: Figure 1 provides an overview of phosphorothioate mutagenesis and summarizes key steps in the process.

1. Generation of a Library of Mutants — Doping DNA Template with Non-wild Type Nucleotides

1. Synthesize T7 Primer (5’-GTAATACGACTCACTATAG-3’) by chemical synthesis on a DNA synthesizer.
2. Synthesize a doped oligonucleotide (complementary strand) by chemical synthesis on a DNA synthesizer corresponding to the protein binding site. Use an appropriate mixture of phosphoramidites during chemical synthesis for each site of doping (X below) with a ratio of 90% A as the wild-....

Results

Principle of saturation mutagenesis using doping:

For an appropriate molar ratio of wild-type and other nucleotides, use an equal mixture of all four nucleotides if only one position is to be analyzed. However, if multiple positions are analyzed simultaneously, the ratio of non-wild type to wild- type nucleotides must be adjusted, i.e., reduced. Otherwise, in addition to single substitutions, which is desired, there will a.......

Discussion

Mutagenesis has long been used to characterize protein binding sites. First, a series of mutants can be constructed and individually tested in binding assays to analyze their effects on binding affinity. While a standard mutagenesis approach offers a way to analyze several sequences, multiple steps involved in the standard approach, such as constructing mutants and performing a series of binding reactions for each mutant, is laborious and time consuming and may not allow saturation mutagenesis, especially for longer sequ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Uridine 5’ a-thio triphosphate	NEN (Boston, Massachusetts)	NLP-017
Adenosine 5’ a-thio triphosphate	NEN (Boston, Massachusetts)	NLP-016
Vacuum manifold	Fisher Scientific	XX1002500	Millipore 25 mm Glass Microanalysis Vacuum Filter
Vacuum manifold	Millipore	XX2702552	1225 Sampling Vacuum Manifold
Nitrocellulose	Millipore	HAWP
Nitrocellulose	Schleicher & Schuell	PROTRAN
Dephosphorlyation Kit	NEB	M0508
T4 Polynucleotide Kinase	NEB	M0201S
Proteinase K	NEB	P8107S
T7 RNA polymerase	NEB	M0251S
RNasin	Promega		RNase inhibitor
Glass Plates	Standard	Standard
Gel Electrophoresis equipment	Standard	Standard
X-ray films	Standard	Standard
Polyacrylamide gel solutions	Standard	Standard