Reconstruction of translation

Abstract

The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature-sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read-through of nonsense codons, occurs. Using reversal of temperature-sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain. Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70S ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43 degrees C) and non-restrictive (35 degrees C) temperatures. Fewer polysomes and a larger proportion of 70S ribosomes relative to subunits were observed at 43 degrees C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35 degrees C with either strain, but restored the polysome pattern of N4316 at 43 degrees C. The purified rescue protein labelled by methylation retained activity and bound preferentially to 30S subunits. Rescue bound to 30S particles prevented the action of IF-3 fostering formation of 70S ribosomes. Thus the rescue protein enables formation of 70S ribosomes from 30S and 50S subunits. 70S ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70S ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation.