In Vivo Ribosome-Amplified MetaBOlism, RAMBO, Effect Observed by Real Time Pulse Chase, RTPC, NMR Spectroscopy.

Quinary interactions between proteins and ribosomes play an important role in regulating biological activity through a phenomenon termed the Ribosome-Amplified MetaBOlism, RAMBO, effect. This effect has been documented in vitro but not in vivo. Real time pulse chase, RTPC, NMR spectroscopy, coupled with isotopic flux analysis in Escherichia coli was used to validate the RAMBO effect in vivo. The ribosomal-targeting antibiotic chloramphenicol was employed to disrupt the quinary structure of pyruvate kinase, the final enzyme in glycolysis. Kinetic flux profiling demonstrated that the in vitro deactivation of the RAMBO effect by chloramphenicol was also observed in vivo, thereby confirming the potential role of ribosomes in regulating glycolysis. The noninvasive modular design of the RTPC-NMR platform allows for high-resolution metabolic monitoring across different cell types, providing broad applicability for studying the real-time metabolic responses to external stimuli in living cells.