Ribosomes transit between two conformational states, non-rotated and rotated, through the elongation cycle. Here, we present evidence that an internal loop in the essential yeast ribosomal protein rpL10 is a central controller of this process. Mutations in this loop promote opposing effects on the natural equilibrium between these two extreme conformational states. rRNA chemical modification analyses reveals allosteric interactions involved in coordinating intersubunit rotation originating from rpL10 in the core of the large subunit (LSU) through both subunits, linking all the functional centers of the ribosome. Mutations promoting rotational disequilibria showed catalytic, biochemical and translational fidelity defects. An rpL3 mutation promoting opposing structural and biochemical effects, suppressed an rpL10 mutant, re-establishing rotational equilibrium. The rpL10 loop is also involved in Sdo1p recruitment, suggesting that rotational status is important for ensuring late-stage maturation of the LSU, supporting a model in which pre-60S subunits undergo a ‘test drive’ before final maturation.
CD200:CD200R1 plays an immunoregulatory role in vivo.
cThe Pea enation mosaic virus (PEMV) 3= translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5=-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5= and 3= PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3= translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2=-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5= hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5= end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation. C ellular gene expression is regulated at multiple steps, including when mRNAs are translated into proteins. Posttranscriptional control of gene expression includes events that occur during translation initiation, when the correct start codon is identified and decoded (1, 2). Canonical translation initiation in eukaryotes is a complex, multistep process in which the 5= m7GpppN cap and 3= poly(A) tail cooperate to recruit translation initiation factors and ribosomal subunits. The 5= cap is recognized by the cap-binding protein eIF4E, which along with the scaffold protein eIF4G is one subunit of the eukaryotic translation initiation factor complex eIF4F (3). Simultaneous binding of eIF4G to eIF4E and poly(A)-binding protein forms a bridge that circularizes the mRNA, which is thought to recycle ribosomes, leading to more efficient translation (4). The 43S ribosome preinitiation complex (PIC), consisting of the 40S ribosomal subunit-and Met-tRNA-containing ternary complex, is recruited to the 5= end of mRNA via the interaction between eIF4G and additional initiation factors (3, 5). The PIC then scans the message in a 5=-to-3= direction until contacting an initiation codon in the proper context. The 60S ribosomal subunit joins to form the 8...
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