Targeting Translation Activity at the Ribosome Interface with UV-Active Small Molecules

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Puromycin is a standard antibiotic for studying protein synthesis and ribosome activity by incorporating into nascent peptides.
• The effects of puromycin beyond the ribosome's catalytic core are not well understood.
• Understanding small molecule interactions with ribosome-associated proteins is crucial for elucidating translational regulation.

Purpose of the Study:

• To develop novel puromycin analogues that interact with ribosome-associated proteins.
• To map direct small molecule-protein interactions in living cells.
• To explore new methods for sensing protein synthesis activity and capturing associated RNA.

Main Methods:

• Development of two tyrosylated-tRNA analogues (3PB and 3PC).
• Biochemical characterization of analogue binding.
• Global mapping of small molecule-protein interactions using clickable and photoreactive probes with mass spectrometry.
• Integration with genome-wide RNA sequencing.

Main Results:

• Identified novel proteins, such as GRP78, directly targeted by puromycin analogues during ribosome activity.
• Demonstrated the potential of these probes to monitor protein synthesis.
• Showcased the ability of the probes to capture associated RNA.
• Established a methodology for exploring unexplored translational control mechanisms.

Conclusions:

• Puromycin analogues can interact with proteins outside the ribosome's catalytic core.
• These novel probes provide powerful tools for investigating ribosome-associated protein interactions and translational regulation.
• The developed methods facilitate the characterization of previously unknown translational control mechanisms.