Elongation factor Tu's nucleotide binding is governed by a thermodynamic landscape unique among bacterial translation factors.

Molecular switches such as GTPases are powerful devices turning "on" or "off" biomolecular processes at the core of critical biological pathways. To develop molecular switches de novo, an intimate understanding of how they function is required. Here we investigate the thermodynamic parameters that define the nucleotide-dependent switch mechanism of elongation factor (EF) Tu as a prototypical molecular switch. EF-Tu alternates between GTP- and GDP-bound conformations during its functional cycle, representing the "on" and "off' states, respectively. We report for the first time that the activation barriers for nucleotide association are the same for both nucleotides, suggesting a guanosine nucleoside or ribose-first mechanism for nucleotide association. Additionally, molecular dynamics (MD) simulations indicate that enthalpic stabilization of GDP binding compared to GTP binding originates in the backbone hydrogen bonding network of EF-Tu. In contrast, binding of GTP to EF-Tu is entropically driven by the liberation of bound water during the GDP- to GTP-bound transition. GDP binding to the apo conformation of EF-Tu is both enthalpically and entropically favored, a feature unique among translational GTPases. This indicates that the apo conformation does not resemble the GDP-bound state. Finally, we show that antibiotics and single amino acid substitutions can be used to target specific structural elements in EF-Tu to redesign the thermodynamic landscape. These findings demonstrate how, through evolution, EF-Tu has fine-tuned the structural and dynamic features that define nucleotide binding, providing insight into how altering these properties could be exploited for protein engineering

Loss of m(1)acp(3)Ψ ribosomal RNA modification is a major feature of cancer

The ribosome is an RNA-protein complex that is essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We identify a cancer-specific single-nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of patients with colorectal carcinoma (CRC) and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine (m(1)acp(3)Ψ), a >1-billion-years-conserved RNA modification at the peptidyl decoding site of the ribosome. A subset of CRC tumors we call hypo-m(1)acp(3)Ψ shows sub-stoichiometric m(1)acp(3)Ψ modification, unlike normal control tissues. An m(1)acp(3)Ψ knockout model and hypo-m(1)acp(3)Ψ patient tumors share a translational signature characterized by highly abundant ribosomal proteins. Thus, m(1)acp(3)Ψ-deficient rRNA forms an uncharacterized class of "onco-ribosome" which may serve as a chemotherapeutic target for treating cancer patients