The Escherichia coli relBE operon encodes a toxin-antitoxin pair, RelE-RelB. RelB can reverse inhibition of protein synthesis by RelE in vivo. We have found that although RelE does not degrade free RNA, it cleaves mRNA in the ribosomal A site with high codon specificity. Among stop codons UAG is cleaved with fast, UAA intermediate and UGA slow rate, while UCG and CAG are cleaved most rapidly among sense codons. We suggest that inhibition of protein synthesis by RelE is reversed with the help of tmRNA, and that RelE plays a regulatory role in bacteria during adaptation to poor growth conditions.

Proteins are made from 19 aa and, curiously, one N-alkylamino acid (''imino acid''), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA Phe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA Pro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the ''tRNA abundance'' hypothesis.non-natural amino acids ͉ ribosomes ͉ tRNA ͉ EF-Tu ͉ GTPase

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (k cat /K M ) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

The first attempt to explain the role of RF3 in translation 2 Corresponding author termination proposed a dual function, first to stimulate the email: ehrenberg@xray.bmc.vv.se binding of RF1 and RF2 (RF1/2) to the ribosome, and secondly to catalyse the dissociation of the factors from Ribosomes complexed with synthetic mRNA and pepthe ribosome after hydrolysis of peptidyl-tRNA in a tidyl-tRNA, ready for peptide release, were purified by reaction dependent on guanine nucleotide, thereby stimugel filtration and used to study the function of release lating the recycling of RF1/2 between ribosomes factor RF3 and guanine nucleotides in the termination (Goldstein and Caskey, 1970). of protein synthesis. The peptide-releasing activity ofRecently, an extended version of the first action of RF3 RF1 and RF2 in limiting concentrations was stimulated proposed by Goldstein and Caskey (1970) has been by the addition of RF3 and GTP, stimulated, though strongly propagated ; Nakamura et al., to a lesser extent, by RF3 and a non-hydrolysable GTP 1996): the 'ternary complex model'. This proposal was analogue, and inhibited by RF3 and GDP or RF3 inspired by the recent discovery of structural similarity without guanine nucleotide. With short incubation between EF-G on one hand and the ternary complex times allowing only a single catalytic cycle of RF1 or between EF-Tu·GTP and aminoacyl-tRNA on the other RF2, peptide release activity was independent of RF3 (AEvarsson et al., 1994;Czworkowski et al., 1994; Nissen and guanine nucleotide. RF3 hydrolysis of GTP to et al., 1995). Domain 4 in EF-G structurally mimics the GDP ⍣ P i was dependent only on ribosomes and not anticodon stem in tRNA, and domains I (the G-domain) on RF1 or RF2. RF3 affected neither the rate of and II in EF-G have sequence similarity with EF-Tu. The association of RF1 and RF2 with the ribosome nor the model was supported further by sequence similarities catalytic rate of peptide release. A model is proposed between RF3 on one hand and EF-Tu and EF-G on the which explains how RF3 recycles RF1 and RF2 by other, as well as by sequence similarities between the displacing the factors from the ribosome after the part of EF-G that mimics the tRNA anticodon stem, and release of peptide.RF1/2. From these data, it was suggested that RF3, GTP Keywords: protein synthesis/release factors/RF3/ and RF1 or RF2 may form a ternary complex like that ribosome/termination between EF-Tu, GTP and aminoacyl-tRNA, either off or on the ribosome (Ito et al

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.