Go Hirokawa

Post‐termination complex disassembly by ribosome recycling factor, a functional tRNA mimic

Ribosome recycling factor (RRF) together with elongation factor G (EF‐G) disassembles the post‐ termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post‐termination complex. In contrast, fusidic acid and a GTP analog that fix EF‐G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF‐G alone. The data are consistent with the notion that RRF binds to the A‐site and is translocated to the P‐site, releasing deacylated tRNA from the P‐ and E‐sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF‐G from the ribosome. With the model post‐termination complex, 70S ribosomes were released from the post‐termination complex by the RRF reaction and were then dissociated into subunits by IF3.

In vivo effect of inactivation of ribosome recycling factor – fate of ribosomes after unscheduled translation downstream of open reading frame

The post‐termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non‐permissive temperature of a temperature‐sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3′ end of mRNA. However, instead of accumulating at the 3′ end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer‐messenger RNA from the 3′ end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis.

Inhibition of antiassociation activity of translation initiation factor 3 by paromomycin

ABSTRACT The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg2+. Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.

CRYSTALLIZATION AND PRELIMINARY X-RAY ANALYSIS OF THERMOTOGA MARITIMA RIBOSOME RECYCLING FACTOR

Thermotoga maritima ribosome recycling factor (RRF) is one of the proteins catalyzing the fourth step in prokaryotic protein synthesis, ribosome recycling. The RRF protein was crystallized with ammonium sulfate. Native diffraction data to 2.55 A resolution were obtained at the MAX II synchrotron from a flash-frozen crystal at 100 K. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 47, c = 298 A, and probably contain one monomer per asymmetric unit.