Biology subjects

Sekulski, K.

Publications and source records attributed to Sekulski, K..

3 recordsLinked to original sources

bioRxiv2022

rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly

Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain (spb1D52A) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF4- transition state structure that shows the direct involvement of unmodified G2922 in Nog2 GTPase activation. Intragenic and dosage suppressors indicate that the biogenesis defect in the spb1D52A strain is caused by reduced initial recruitment of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 stability on the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Because multiple K-loop GTPases are involved in the assembly of ribosomes and other RNPs, our findings provide a template to study the role of RNA trans factors in modulating the regulatory functions of this important family of enzymes.

biophysics↗

bioRxiv2022

Sequence-directed RNA remodeling within a topologically complex RNP substrate

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes is at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-directed remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize a post-catalytic, high-energy intermediate that drives the organization of the root helix structure within rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

biophysics↗

bioRxiv2018

Anatomy of a pressure sensing protein kinase

Cells respond to hydrostatic pressure to maintain cellular, organ, and organism level functions, yet the direct pressure sensors are largely unknown. Here we show that hydrostatic pressure directly activates With No Lysine(K) kinase-3 (WNK3) 1, a soluble intracellular protein kinase. Using gel filtration we demonstrate that pressure induces a dimer to monomer transition in a construct of the unphosphorylated kinase domain of WNK3 (uWNK3-KDm or uWNK3). The uWNK3 has not been crystallized, but crosslinking data suggest that the uWNK3 dimer corresponds to crystallographically observed dimer of WNK1 (uWNK1-KDm, or uWNK1) 2,3. Sequence alignments with WNKs from species living in different pressure environments and mutational analysis lend further support for this idea. Unique features of the uWNK1 structure suggest a mechanism involving bound water. We further show that hydrostatic pressure activates full-length WNK3 in D. melanogaster tubules.

biophysics↗