N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indic ated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form m ay be inactivated without being degraded. These experiments were carried ou t by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-termin al and C-terminal modifications of the coding sequence on the ribosomal rel ease/termiaation process and on the stability of the newly synthesized prot ein. Elimination;of the leader sequence in two proteins tested, mitochondri al rhodanese and bacterial p-lactamase, caused an increase in the percentag e of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-tag impaired the ri bosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribos omes. A C-terminal extension of the coding sequence for rhodanese by one am ino acid decreased the percentage released polypeptide and furthermore affe cted the stability of the in vitro formed protein. We propose that a regula tory mechanism exists by which N-terminal and C-terminal sequences of a new ly synthesized protein have feed-back effects on the termination factor-med iated release and on the stability of the native three-dimensional structur e. (C) 1999 Elsevier Science Ltd. All rights reserved.