Hidden specificity in an apparently nonspecific RNA-binding protein

• Published in: Nature (London) Vol. 502; no. 7471; pp. 385 - 388
• Main Authors: Guenther, Ulf-Peter, Yandek, Lindsay E., Niland, Courtney N., Campbell, Frank E., Anderson, David, Anderson, Vernon E., Harris, Michael E., Jankowsky, Eckhard
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.10.2013; Nature Publishing Group
• Subjects: 5' Untranslated Regions - genetics; 631/45/147; 631/45/173; 631/45/500; Base Sequence; Binding proteins; Binding sites; Deoxyribonucleic acid; DNA; DNA binding proteins; E coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Genetic aspects; Genetic research; Humanities and Social Sciences; Kinetics; Leadership; letter; multidisciplinary; Nucleic Acid Conformation; Nucleic acids; Physiological aspects; Protein binding; Proteins; Ribonuclease P - chemistry; Ribonuclease P - genetics; Ribonuclease P - metabolism; RNA; RNA Precursors - chemistry; RNA Precursors - genetics; RNA Precursors - metabolism; RNA processing; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; RNA, Transfer, Met - chemistry; RNA, Transfer, Met - genetics; RNA, Transfer, Met - metabolism; Science; Signatures; Substrate Specificity; Substrates; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature12543

A novel high-throughput sequencing kinetics approach is used to measure functional binding of the apparently nonspecific RNA-binding protein C5 to all possible sequence variants in its substrate binding site; C5 binds different substrate variants with affinities varying widely, and with a similar affinity distribution to that of highly specific nucleic-acid-binding proteins, but it does not bind its physiological RNA targets with the highest affinity. A specificity to nonspecificity spectrum The interaction of 'specific' binding proteins with nucleic acids is attributed to high-affinity interaction with favoured sequences or structures. Proteins that lack recognized cognate sites are considered to bind 'non-specifically'. This paper presents a new and unexpected view on the concept of nonspecificity/specificity for nucleic acid-binding proteins. Eckhard Jankowsky and colleagues show that the nonspecific RNA-binding protein, C5, which is a subunit of RNase P, exhibits an underlying specificity. Using a novel high-throughput kinetic approach, they measure affinities of C5 for different sequences that vary over several orders of magnitude. Compared with specific binding protein affinities, C5 binds with affinities that are more moderate, suggesting that the only distinction between specific and nonspecific binding proteins is the region of the affinity distribution that is used. Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA 1 , 2 . Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both 1 , 2 , 3 , 4 . Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific 1 , 2 , 5 . Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures 6 . Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli . The protein binds 5′ leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.