A direct estimation of the context effect on the efficiency of termination

An in vitro assay in which terminating Escherichia coli ribosomes with diff erent stop signals in the A-site compete for a limited amount of a release factor (RF1 or RF2) has been used to estimate the relative termination effi ciencies at stop codons with different adjacent downstream nucleotides. The assay allows direct measurements of relative k(cat)/K-m parameters for the productive association of release factors to ribosomes. The k(cat)/K-m par ameter is larger for UAA(U) than for UAA(C) programmed ribosomes and the di fference in k(cat)/K-m is much larger for RF2 (about 80%) than for RF1 (abo ut 30%). These differences in the k(cat)/K-m parameter are not affected by the addition of release factor RF3. The only discernible effect of RF3 is a considerable acceleration of RF1/2 recycling. The estimated k(cat)/K-m parameters correlate well with the affinities of r elease factors for ribosomes programmed with different stop signals. These affinities were estimated from the extent of inhibition of ribosomal recycl ing by high concentrations of release factors in the absence of release fac tor RF3. The affinity for RF2 depends on the immediate downstream context o f the stop codon in the translated mRNA and is about three times higher for UAA(U) than for UAA(C). The corresponding difference in affinities for RF1 is twofold. For all stop signals studied, the estimated affinity of RF2 fo r terminating ribosomes is much lower than that of RF1. It is also striking that the affinity of ribosomes for a chromosomally expressed RF2 is at lea st three times higher than for RF2 isolated from an overproducing E. coli s train. (C) 1998 Academic Press.