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- Introduction
- Real-time PCR
- Microarrays
- What is Next Generation DNA Sequencing?
- RNA sequencing
- Biological interpretation of gene expression data
- Genotyping, epigenetic and DNA/RNA-protein interaction methods
- DNA/RNA-protein interactions
- Summary
- Quiz: Check your learning
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- References
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DNA/RNA-protein interactions
DNA-protein and RNA-protein interactions can have a profound effect on gene expression and the spatial and temporal localisation of mRNA within a cell.
DNA-protein interactions include those between DNA and transcription factors or other regulatory proteins. RNA-protein interactions include those between RNA and the ribosome, and other RNA-binding proteins (21,22).
The analysis of DNA-protein and RNA-protein interactions uses similar methods which are based on (Figure 16):
- immunoprecipitation of the nucleic acid-protein complex
- purification and analysis of the DNA/RNA by microarray or NGS to identify the regions of the genome or transcriptome that are interacting with a specific protein at a specific point in time (23)
DNA-protein analyses are referred to as ChIP-chip or ChIP-seq, and require the crosslinking of DNA-protein with formaldehyde before the immunoprecipitation.
For RNA-protein analyses the cross-linking step is optional. RNA-immunoprecipitation (RIP)-chip/seq analyses can be performed with or without formaldehyde-based cross-linking before immunoprecipitation. Without cross-linking, this method is not well suited to detecting transient RNA-protein interactions and is prone to high background. Cross-linking and immunoprecipitation (CLIP)-chip/seq analyses use ultra violet cross-linking of RNA and proteins before immunoprecipitation, thus overcoming many of the limitations of RIP-based methods (21,23,24).