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 been constructed to study the role of the release factors in termination, in particular whether each possesses specific domains for recognition of the stop codon, and for facilitating peptidyl-tRNA hydrolysis. One hybrid factor showed normal codon-recognition activity but was defective in its ability to facilitate hydrolysis. Overexpression of this protein was toxic to the cell. Conversely, another hybrid factor showed complete loss of codon recognition but retained some hydrolysis activity. These two functional activities of the release factors were not localised in domains within either the amino-terminal or carboxy-terminal halves of the primary sequence as previously predicted. Evidence from the activities of the hybrid proteins and from earlier studies suggests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, contribute to the hydrolysis domain, whereas residues from both the amino-terminal and carboxy-terminal halves of the molecule are important for the codon recognition domain.
Moffat
J G
JG
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Donly
B C
BC
McCaughan
K K
KK
Tate
W P
WP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010454:Peptide Termination Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D009838:Oligodeoxyribonucleotides; D010454:Peptide Termination Factors; D011993:Recombinant Fusion Proteins; C041460:peptide chain termination release factor 2