David V. Freistroffer

Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP‐dependent manner

Ribosomes complexed with synthetic mRNA and peptidyl‐tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide‐releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non‐hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + Pi was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl‐tRNA synthetases. In this system, ribosomes perform repeated rounds of translation of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured. The system has been used to study the influence of release factor RF3 and of ribosome recycling factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is ∼40 s. This time is reduced to ∼30 s by the addition of RF3 alone and to ∼15 s by the addition of RRF alone. When both RF3 and RRF are added to the translation system, the recycling time drops to <6 s. Release factor RF3 is seen to promote RF1 cycling between different ribosomes. The action of RRF is shown to depend on the concentration of elongation factor‐G. Even in the presence of RRF, ribosomes do not leave the mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome. It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.

Mutations in RNAs of both ribosomal subunits cause defects in translation termination

Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl‐tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon‐dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants. Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl‐tRNA hydrolysis during termination of protein biosynthesis. The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution. Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination.

Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs.

A method to synthesise region-labelled proteins for structural studies with NMR is suggested. The technique is based on in vitro translation of matrix-coupled mRNAs. Translation starts with unlabelled amino acids from the initiation codon of the mRNA and continues to the beginning of the region of interest. Here, the ribosomes pause while the tRNAs charged with unlabelled amino acids are replaced with tRNAs charged with isotope-labelled amino acids. Translation then proceeds through the region of interest until the ribosomes pause at its end. At this point aminoacyl-tRNAs are changed again. Translation is resumed with unlabelled amino acids and continues to the STOP codon of the mRNA, where the ribosomes pause. In the final step the complete, region-labelled protein is eluted from the column in almost pure form. The method is demonstrated for small scale synthesis of the DNA binding domain (DBD) of the glucocorticoid receptor (GR), where the DNA-recognising helix is labelled but the rest of DBD is unlabelled. The new technique can be generalised to allow a desired region in a protein to be isotope-labelled.

PURIFICATION OF ACTIVE ESCHERICHIA COLI RIBOSOME RECYCLING FACTOR (RRF) FROM AN OSMO-REGULATED EXPRESSION SYSTEM

Ribosome release factor (RRF) from Escherichia coli was overproduced from an osmo-expression vector. More than 40% of cell protein was RRF after 6 h of induction. A purification scheme is described that produced 50 mg of RRF from an initial culture of 2 L. The recycling time for ribosomes synthesising the tripeptide fMet-Phe-Leu in vitro in the absence of RF3 was reduced from 40 to 15 s by the addition of purified 1.5 microM RRF.