The Concentration of Polypeptide Chain Release Factors 1 and 2 at Different Growth Rates of Escherichia coli

Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance

“…Analysis at the protein level showed RF2 expression in E. coli to be 6 -7-fold higher than RF1 expression (Fig. 4A, red bars), which is in good agreement with previous studies (19,20). Thus, the RF2:RF1 ratio matched well with the ratio of TGA to TAG.…”

“…3). Earlier, the low abundance of TAG was speculated to correlate with lower efficiency of termination with RF1 (14,19,20). When compared for k cat /K m , which is often correlated with efficiency in an enzymatic reaction, the value reported for RF1 is 6.0 ϫ 10 7 , and that for RF2 (Chromosomal) is 4.6 ϫ 10 7 M Ϫ1 s Ϫ1 , which apparently implies that RF1 has about 1.5 times higher efficiency than RF2 (27).…”

“…Previous results showed that in exponentially growing E. coli RF2 is about 5 times more abundant than RF1 (19,20). In the present study, we analyzed three model bacteria, E. coli, M. smegmatis, and B. subtilis, for RF1 and RF2 abundance using qPCR and Western blotting.…”

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

“…These observations are consistent with a model in which inefficient translation termination at the ybeL stop codon permits SsrA to compete with RF-1 and RF-2 for entry into the ribosomal A-site, leading to higher levels of full-length protein tagging. The RF-1 and RF-2 proteins are present in ϳ10-fold and 50-fold excess, respectively, over SsrA RNA in E. coli during log-phase growth (28). The affinities of these protein release factors for the A-site depends on the identities of the stop codon and following nucleotide (29).…”

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

“…Release factors RF1 and RF2 were purified from wild-type strains (Tate & Caskey, 1990) or from release factor overexpressing strains (Adamski et al+, 1994)+ RF3 was purified as described (Grentzmann et al+, 1994) by HPLC on DEAE (Waters)+ Contamination by elongation factors was efficiently eliminated by a supplementary step on CM-Sephadex (Pharmacia) Grentzmann et al+, 1994)+ Release factor fractions were checked for GTP contamination using electrospray mass spectrometry (Crain, 1990;Straub & Voyksner, 1993)+ Concentrations of purified release factor fractions in terms of amount of protein per unit of volume was not constant compared to the number of molecules of active protein per unit of volume+ For example, overexpression of release factors has been shown to result in loss of specific activity (Adamski et al+, 1994) and a possible site for posttranslational modification for RF2 has been reported (Uno et al+, 1996)+ We therefore estimated the quantities of active release factors in units of activity as described previously (Caskey et al+, 1971;Grentzmann et al+, 1994)+ tRNAfmet (Subriden) was charged and purified according to Tate and Caskey (1990)+ Tight couple ribosomes were purified as described (Spedding, 1990)+ UUC AUG UAA, UUC AUG UAG, and UUC AUG UGA RNA oligonucleotides were synthesized on an ABI synthesizer, deprotected, and purified on Sephadex G-25 (Pharmacia)+ Sequences were verified by mass spectroscopy (Limbach et al+, 1995)+ RRF was purified from an overexpressing strain (Ichikawa & Kaji, 1989) as described (Hirashima & Kaji, 1972)+ EF-G was purified and its activity was tested as described (Kaziro et al+, 1972)+…”