Post Translational Addition of Amino Acids - Mechanisms Substrate Recognition

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Protein degradation is fundamental to a variety of cellular processes which include gene regulation, attenuating of proteolytic cascades and the removal of mis-folded or damaged proteins. We are investigating the evolutionary conserved degradation signal known as the N-degron, which is the signal for N-End Rule degradation in both eubacteria and eukaryotes. The N-end rule pathway is a ubiquitous process that targets proteins for degradation that possess a particular N-terminal amino acid. Aminoacyl-tRNA protein transferases catalyze the post-translational addition of destabilizing amino acids from aminoacyl-tRNAs to the N-termini of substrate proteins, targeting them for N-End rule degradation. To investigate the activity of the bacterial aminoacyl-tRNA protein transferase, L/F transferase, we have recently developed a novel mass spectrometry based procedure to quantify enzymatic activity. With the available X-ray structural data available for L/F transferase and out mass spectrometry method we have completed investigations that have enabled us to propose a new catalytic mechanism for the enzyme. We propose to continue our work on L/F transferase and focus on substrate recognition. Recently the first in vivo L/F transferase substrate was identified and remarkably this substrate lacks the characteristic N-termini that would predicted from historic biochemical data on L/F transferase. This discrepancy of biochemical data and recent in vivo data argues for a systematic evaluation of sequence requirements for substrate recognition and the additional identification of substrate proteins. With modern mass spectrometry techniques we will comprehensively investigate the substrate sequence selectivity of E. coli L/F transferase and use 'click chemistry' and proteomic approaches to identify additional in vivo L/F transferase substrates. We will also broaden our focused investigations on L/F transferase to examine aspects of protein turnover as a whole in E. coli.