RESUMEN
Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/genética , Precursores del ARN/química , Ribonucleasa P/química , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5' end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP-pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4-5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5' leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP-substrate recognition and provides a new predictive model for the PRORP-substrate complex.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Precursores del ARN/metabolismo , ARN de Planta/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Conformación de Ácido Nucleico , Precursores del ARN/química , Precursores del ARN/genética , ARN de Planta/química , ARN de Planta/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Ribonucleasa P/química , Ribonucleasa P/genéticaRESUMEN
Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5' end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5' maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field.
