Ram ribosomes are defective proofreaders

Abstract

We have studied the kinetics of poly(U) translation by three ribosomal ambiguity (Ram) mutants in an in vitro system with performance characteristics similar to those expressed in vivo. The leucine missense frequency supported by Ram ribosomes with tRNA ₂ ^Leu increases between six and twelve-fold over that of wild-type ribosomes, while the corresponding increase with tRNA ₄ ^Leu was between four and eight-fold, depending on the rpsD allele. We have used a steady-state assay for profreading to identify the kinetic lesion responsible for the Ram phenotype. We were unable to detect any difference between Ram and wild-type ribosomes with respect to the initial kinetics of amino-acyl-tRNA selection. All of the increased error rates could be associated with a decreased capacity of these Ram ribosomes to discard non-cognate aminoacyl-tRNA by proof reading.