Analysis of the RNA Binding Specificity Landscape of C5 Protein Reveals Structure and Sequence Preferences that Direct RNase P Specificity

Abstract

Summary RNA-binding proteins (RBPs) are typically involved in non-equilibrium cellular processes, and specificity can arise from differences in ground state, transition state or product states of the binding reactions for alternative RNAs. Here, we use high throughput methods to measure and analyze the RNA association kinetics and equilibrium binding affinity for all possible sequence combinations in precursor tRNA binding site of C5, the essential protein subunit of Escherichia coli ribonuclease P. The results show that the RNA sequence specificity of C5 arises due to favorable RNA-protein interactions that stabilize the transition state for association and bound ES complex. Specificity is further impacted by unfavorable RNA structure involving the C5 binding site in the ground state. The results illustrate a comprehensive quantitative approach for analysis of RNA binding specificity, and show how both RNA structure and sequence preferences of an essential protein subunit direct the specificity of a ribonucleoprotein enzyme.

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

“…2) (11). Bacterial RNase P is a ribonucleprotein enzyme that removes 5′ leader sequences from pre-tRNAs (12).…”

“…To account for the fact that inherent RBP specificity can arise from discrimination between substrate variants in ground and transition states (Fig. 1), we have recently expanded the HiTS-Kin approach by a complementary technique, HiTS-EQ (11). The combination of the HiTS-Kin and HiTS-EQ approaches allows assessment and, to a large extent, deconvolution of RBP discrimination in ground and transition state (11).…”

“…1), we have recently expanded the HiTS-Kin approach by a complementary technique, HiTS-EQ (11). The combination of the HiTS-Kin and HiTS-EQ approaches allows assessment and, to a large extent, deconvolution of RBP discrimination in ground and transition state (11). The extent of substrate discrimination at different reaction stages the is called the RBP specificity landscape (11).…”

The DEAD-box RNA helicases and multiple abiotic stresses in plants: a systematic review of recent advances and challenges

“…Molecular processes such as replication, transcription, posttranscriptional modification, translation, mRNA splicing and mRNA export and its stability are vital for living organisms, and helicase enzymes are to play very crucial role in gene expression and regulation (Hartman et al, 2006;Bolinger et al, 2007;Bolinger et al, 2010;Górna et al, 2010;Ranji et al, 2011;Andreou and Klostermeier, 2013;Chu et al, 2016;Lin et al, 2016;Padmanabhan et al, 2016). Helicase enzymes are categorized into two types namely, DNA helicase and RNA helicase (Jankowsky, 2011).…”

RNA Binding: Getting Specific about Specificity

“…On page XXX of this issue of Cell Chemical Biology , Lin et al (2016) demonstrate how to employ high-throughput sequencing methods to determine the K a of the C5 protein of ribonuclease P (RNase P) for all possible leader sequences of pre-tRNA. RNase P is the enzyme responsible for cleaving the leader sequence present in pre-tRNAs, thereby producing their mature 5'-termini.…”