K Yoshimura1, K Ito, Y Nakamura. 1. Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Abstract
BACKGROUND: Prokaryotic translational release factors, RF1 and RF2, catalyse protein release at UAG/UAA and UGA/UAA stop codons, respectively. Mutations in RF1 and RF2 are known to cause non-sense suppression for UAG (amber) and UGA (opal) codons, respectively, and they do not exert a reciprocal ('cross') suppression phenotype. We aimed to isolate RF mutants of such cross-suppression activity, which we designated 'Csu' phenotype in this paper. RESULTS: Using a lacZ (UAG) reporter, we selected amber suppressor alleles occurring in the plasmid-bearing RF2 gene of Salmonella typhimurium. Of nine such RF2 csu alleles, five were mis-sense mutations and four were non-sense mutations. The former mis-sense mutants retained the RF2 activity and catalysed UGA termination both in vivo and in vitro. RF2 C-terminal deletions equivalent to the non-sense alleles exerted amber suppression as well as opal suppression activity. Moreover, the equivalent RF1 segments also showed both the suppression phenotypes. CONCLUSIONS: All the csu mutations were mapped at the C-terminal half of RF2 and are strikingly coincident with the highly conservative amino acids, suggesting that they affect the conserved function of bacterial RFs. We propose here that there should be an 'initial binding' step of RFs to the ribosome, preceding stop codon recognition ('initial binding' hypothesis) and that the N-terminal RF domain(s), that are truncated or affected by the csu mutations, are responsible for this step and interfere with the proper functioning of cognate release factors on the ribosome.
BACKGROUND: Prokaryotic translational release factors, RF1 and RF2, catalyse protein release at UAG/UAA and UGA/UAA stop codons, respectively. Mutations in RF1 and RF2 are known to cause non-sense suppression for UAG (amber) and UGA (opal) codons, respectively, and they do not exert a reciprocal ('cross') suppression phenotype. We aimed to isolate RF mutants of such cross-suppression activity, which we designated 'Csu' phenotype in this paper. RESULTS: Using a lacZ (UAG) reporter, we selected amber suppressor alleles occurring in the plasmid-bearing RF2 gene of Salmonella typhimurium. Of nine such RF2 csu alleles, five were mis-sense mutations and four were non-sense mutations. The former mis-sense mutants retained the RF2 activity and catalysed UGA termination both in vivo and in vitro. RF2 C-terminal deletions equivalent to the non-sense alleles exerted amber suppression as well as opal suppression activity. Moreover, the equivalent RF1 segments also showed both the suppression phenotypes. CONCLUSIONS: All the csu mutations were mapped at the C-terminal half of RF2 and are strikingly coincident with the highly conservative amino acids, suggesting that they affect the conserved function of bacterial RFs. We propose here that there should be an 'initial binding' step of RFs to the ribosome, preceding stop codon recognition ('initial binding' hypothesis) and that the N-terminal RF domain(s), that are truncated or affected by the csu mutations, are responsible for this step and interfere with the proper functioning of cognate release factors on the ribosome.