Translational control 1995-2015: unveiling molecular underpinnings and roles in human biology.

The last 20 years have seen tremendous progress in deciphering the fundamental mechanisms of protein synthesis and molecular pathways of translational control, and in implicating these processes in development, neurobiology, and etiologyof human diseases. Muchof this success can be attributed to the introduction of new technologies, such as next-generation RNA sequencing and advances in cryo-EM; and also to development of powerful in vitro systems recapitulating complex in vivo phenomena with purified components. Also critical have been strategic interactions between experts in different disciplines of biology, such as the wedding of biochemistry with genetics and structural biology, and of translation biochemistry with neurobiology and medicine. RNA has contributed to this progress by publishing key findings on molecular mechanisms of protein synthesis and the role of translational control in regulating gene expression in diverse organisms. Thebasic reactions of the elongationand termination stages of protein synthesis are highly conserved throughout all three kingdoms of life. Because they occur in the catalytic center of the ribosome, their molecular mechanisms were illuminated by the stunning successes of X-ray crystallography and cryo-electron microscopy in producing high-resolution structures of both empty ribosomes and a myriad of ribosome complexes with mRNA, tRNAs, or elongation, termination, or ribosome recycling factors trapped in different stages of the process. Recent innovations in cryo-EM technology are revealing ribosome structures at nearly 3 A resolution; and new algorithms for sorting structurally distinct molecules means that sample heterogeneity (an anathema to crystallography) is now exploited to identify different conformational states in the same preparation. The solved ribosome structures revealed the path of mRNA and the A, P, and E binding sites for the tRNA, as it progress through the elongation cycle from aminoacyltRNA, to peptidyl-tRNA to deacylated tRNA, located at the interface between the large and small subunits; as well as the peptidyl transferase center wherepeptide bond formation occurs. As the decoding center of the ribosome is comprised almost entirely of rRNA, it became clear that the ribosome is a ribozyme. The protein factors involved in translocation of mRNA and the tRNAs through the decoding sites (EF-G/ EF2), in recognizing stop codons and hydrolyzing peptidyltRNA (RF1/eRF1), and splitting apart ribosomal subunits following termination (bacterial RRF) all exploit striking structural mimicry of tRNA to fit into the A site. Interestingly, RF1 was shown to contain a protein element that “reads” the stop codon and another that reaches into the peptidyl transferase center to hydrolyze the peptidyl-tRNA linkage. The peptidyl-tRNA and aminoacyl-tRNA are anchored in the precise locations required for peptide bond formation, but the ribosome is not simply a rigid catalytic platform, but undergoes conformational changes in selecting the correct EF1A·GTP·aminoacyl-tRNA ternary complex for each codon, and displays inter-subunit ratcheting during translocation and termination. GTP binding and hydrolysis by the elongation factors evoke cyclic conformational changes that drive the process forward. The use of fluorescently labeled reactants and stopped-flow kinetic analysis allowed a detailed dissection of different steps in the processes of aminoacyltRNA selection in the A site, with proofreading of the codon:anticodon interaction; and of translocation of tRNAs through the decoding center catalyzed by EF2. Single-molecule approaches also contributed importantly to this progress, by identifying intermediate states too transient to be detected by ensemble kinetic experiments or structural biology. In other developments, eIF5A and its bacterial counterpart EF-P were shown to stimulate elongation specifically through stretches of proline codons, and both factors occupy the E-site and communicate directly with the peptidyl transferase center. Interestingly, RRF is replaced in eukaryotes and archaea by an ATP-binding cassette protein, ABCE1/ Rli1, that becomes wedged between the large and small subunits and uses ATP hydrolysis to split apart the subunits. ABCE1/Rli1also functions with RF1 paralog Dom34 to