FUNCTIONAL DOMAINS IN THE ESCHERICHIA-COLI RELEASE FACTORS - ACTIVITIES OF HYBRIDS BETWEEN RF-1 AND RF-2

Chimeras between Escherichia coli release factors RF-1 and RF-2 have b een constructed to study the role of the release factors in terminatio n, in particular whether each possesses specific domains for recogniti on of the stop codon, and for facilitating peptidyl-tRNA hydrolysis. O ne hybrid factor showed normal codon-recognition activity but was defe ctive in its ability to facilitate hydrolysis. Overexpression of this protein was toxic to the cell. Conversely, another hybrid factor showe d complete loss of codon recognition but retained some hydrolysis acti vity. These two functional activities of the release factors were not localised in domains within either the amino-terminal or carboxy-termi nal halves of the primary sequence as previously predicted. Evidence f rom the activities of the hybrid proteins and from earlier studies sug gests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, c ontribute to the hydrolysis domain, whereas residues from both the ami no-terminal and carboxy-terminal halves of the molecule are important for the codon recognition domain.