Abstract
Author SummaryRibosomes are the cellular machines responsible for all protein synthesis. In eukaryotes, the assembly of ribosomes from their protein and RNA components is extremely complicated and involves more than 200 nonribosomal factors—three times the number of proteins in the mature complex. Among these factors, the Fap7 protein is particularly intriguing because it interacts with the small subunit ribosomal protein Rps14 and it exhibits adenylate kinase activity—a molecular function more commonly associated with regulating ATP/ADP levels than assembling protein–RNA complexes. Combining structural and biochemical analysis of the Rps14–Fap7 complex, we show that Fap7 uses protein side chains to mimic RNA contacts, rendering the interaction of Rps14 with ribosomal RNA or with Fap7 competitive and mutually exclusive. Once bound, Rps14 blocks the substrate-binding cavity of Fap7, and ATP hydrolysis will then break the Fap7–Rps14 complex apart. At the same time, the ribosome structure at the location where Rps14 binds is disrupted when the Fap7/Rps14 complex is formed, and this process is regulated by ATP binding and hydrolysis. Our model is thus that Fap7 temporarily removes Rps14 from the ribosome to enable a conformational change of the ribosomal RNA that is needed for the final maturation step of the small ribosomal subunit.
Highlights
Over 200 preribosomal factors are involved in the maturation of ribosomes
adenylate kinase (AK) catalyze the reversible transfer of the c phosphate of adenosine nucleotide triphosphate (ATP) to an adenosine mono-phosphate (AMP), forming two molecules of adenosine di-phosphate (ADP)
Two crystal forms were obtained for the archaeal aFap7– aRps14 complex bound to ADP/Mg2+ and ATP/Mg2+, which diffracted to 2.1 Aand 2.4 A, respectively
Summary
Over 200 preribosomal factors are involved in the maturation of ribosomes. Most of these factors are essential for cell survival, but their precise molecular functions remain elusive (for reviews, see [1,2,3]). One of the last steps of maturation of the small subunit of the ribosome is the cytoplasmic cleavage of the 20S pre-rRNA at site D to generate 18S rRNA. This cleavage is carried out by the endonuclease Nob in 80S-like complexes composed of pre-40S particles and mature 60S [4,5]. The locations of maturation factors in the late pre-40S particles is emerging from in vivo RNA binding (CRAC), cryo-EM, and crystallographic studies on preribosomal particles [6,7,8,9,10], but detailed understanding of their functions remains limited. AKs play important roles in nucleotide metabolism [13], but the link between this enzymatic activity and ribonucleoprotein (RNP) assembly is enigmatic
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