SUPPRESSION OF TEMPERATURE-SENSITIVE DEFECTS OF POLYPEPTIDE RELEASE FACTORS RF-1 AND RF-2 BY MUTATIONS OR BY AN EXCESS OF RF-3 IN ESCHERICHIA-COLI

The termination of protein synthesis in bacteria requires two codon-sp ecific polypeptide-release factors, RF-1 and RF-2. A third factor, RF- 3, stimulates the RF-1 and RF-2 activities in vitro. To clarify the in vivo role of RF-3 for the RF-2 dependent termination, we isolated and characterized suppressor mutations for the temperature-sensitive RF-2 mutation prfB286. One of the intergenic suppressor mutations, srb-1, acquired an up-promoter alteration in the RF-3 gene, which enhanced th e RF-3 expression four- to fivefold. Consistently a threefold increase in the RF-3 level by a promoter-controlled expression plasmid suppres sed prfB286. On the other hand, a temperature-sensitive mutation in RF -1, prfA1, was suppressed only slightly by the high-level expression o f wild-type RF-3. The RF-3 mutations that suppress prfA1 were isolated and named srn. They were classified into four specific alleles; two e ach in the N and C-terminal regions. These altered RF-3 proteins resto red the RF-1-dependent termination at UAG in prfA1 cells. Moreover, th ey enhanced the RF-2-dependent UGA termination in both wild-type and p rfB286 cells. The termination-stimulating activity of RF-3 was further additively increased by the double sm mutations, suggesting that they affected two distinct protein domains that modulate the termination r eaction. Taking these and other results into consideration, RF-3 is li kely to interact functionally and cooperatively with the release facto rs RF-1 and RF-2 in Escherichia coli. (C) 1996 Academic Press Limited