RESUMEN
Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.
Asunto(s)
Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Estrés Fisiológico , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
We report the synthesis and application of a small molecule probe for carbonic anhydrase (CA) to track holo-CA in cell lysates and live-cell models of zinc dyshomeostasis. The probe displays a 12-fold increase in fluorescence upon binding to bovine CA and also responds to human CA isoforms.