E. coli mutants of RF1 and RF2, in which the universal GGQ motif is changed to GAQ, are slow in peptide release from ribosomes. Other kinetic properties are unchanged, suggesting that the GGQ motif is in contact with the peptidyl-transferase center. Deacylated tRNA terminates protein synthesis codon specifically, indicating that the CCA end of tRNA and the GGQ motif operate similarly. Addition of a mutant factor to a pretermination ribosomal complex stimulates exchange of RF3-bound GDP with free GDP, but binding of GTP to RF3 and GTP hydrolysis requires peptide chain release. Therefore, the sequence of steps during termination of translation is regulated by removal of the polypeptide, an event that might trigger a conformational change in the ribosome.

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.

Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N 5 -methylGln by the S-adenosyl L-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N 5 -methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl L-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1⅐eRF3⅐GTP.

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyltRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyltRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coil. A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor ofa leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene pdC. Two transposon insertions were shown to interrupt the coding sequence of prJC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo. The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.The termination step in protein synthesis requires soluble release factors (RFs) that are at least partly responsible for the recognition of messenger-encoded stop signals (1-3). Ribosomal components, especially rRNA, may also be intimately involved (4), and the detailed mechanism of stopsignal recognition remains poorly understood. This is of particular significance because it has become clear that the reading of certain stop signals plays a role in a variety of cellular regulatory events mediated by stop codon readthrough, frameshifting, or other alternate reading events (5).In Escherichia coli, two codon-specific RFs have been purified and characterized. RH is a protein with a molecular weight of 40,460 and shows specificity for UAG and UAA. A second factor, RF2, with a molecular weight of 41,235 is specific for UAA and UGA. Other factors may, however, be involved in the termination process. A third protein component was described that stimulates the activity of RH and RF2 but appears to lack nonsense codon specificity (6, 7). This component was named RF3. In a termination assay in vitro, RF3 lowers the Km for terminator trinucleotides without altering the Vma,, for release and is thought to act at terminator codon recognition (7). The action of the protein is modified by the presence of guanine nucleotides, which has led to the suggestion that RF3 is a GTP-binding protein. RF3 has been at least partially purified (7) and was described as a protein of 46 kDa (8). The genes encoding RF1 and RF2 have been localized on the E. coli chromosome (9, 10), cloned, and sequenced (10-12)....

SummaryRelease factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro , but these mutants could not complement thermosensitive RF mutants in vivo . None of several mutants with altered Gly residues retained activity in vivo or in vitro . Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.

Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.