The base following stop codons in mammalian genes is strongly biased, suggesting that it might be important for the termination event. This proposal has been tested experimentally both in vivo by using the human type I iodothyronine deiodinase mRNA and the recoding event at the internal UGA codon and in vitro by measuring the ability of each of the 12 possible 4-base stop signals to direct the eukaryotic polypeptide release factor to release a model peptide, formylmethionine, from the ribosome. The internal UGA in the deiodinase mRNA is used as a codon for incorporation of selenocysteine into the protein. Changing the base following this UGA codon affected the ratio of termination to selenocysteine incorporation in vivo at this codon: 1:3 (C or U) and 3:1 (A or G). These UGAN sequences have the same order of efficiency of termination as was found with the in vitro termination assay (4th base: A G> > C U). The efficiency of in vitro termination varied in the same manner over a 70-fold range for the UAAN series and over an 8-fold range for the UGAN and UAGN series. There is a correlation between the strength of the signals and how frequently they occur at natural termination sites. Together these data suggest that the base following the stop codon influences translational termination efficiency as part of a larger termination signal in the expression of mammalian genes.

The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsensesuppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One ofthese mutations, named tos-l (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses ofthe DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-l insertion eliminated the C-terminal one-fifth of the protein.Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codondependent in vitro termination assay. The stimulation was fcantly reduced by GTP, GDP, and the 13,-methylene analog ofGTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designatedpC. A completely nullprJC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions. The termination oftranslation in bacteria requires two codonspecific peptide-chain-release factors: release factor 1 (RF-1; UAG/UAA-specific) and release factor 2 (RF-2; UGA/ UAA-specific) (1). Stop codon recognition by release factors holds considerable interest, since it entails protein-RNA recognition rather than a codon-anticodon (RNA-RNA) interaction, but the mechanism is still unknown (2, 3).The genes encoding the Escherichia coli release factors RF-1 and RF-2 have been isolated (4, 5). The map position of RF-1 (designated prfA) is 27 min on the E. coli chromosome (6), and RF-2 (designated prfB) is at 62 min, within the same operon as the lysyl-tRNA synthetase gene (7). Several mutants of RF-1 and RF-2 have been isolated, and they often cause misreading of stop codons or frameshifting, as well as temperature-sensitive growth of the cells (8-11). Hence the reduced activity of release factors results in several translational errors in vivo, and these errors are probably caused by an abnormally long pausing of ribosomes at stop signals (12).In E. coli, a third factor, RF-3, is known to stimulate the activities of RF-1 and RF-2; it binds guanine nucleotides but is not codon-specific (13,14). This factor has received little attention since its initial characterization. RF-3 was shown to partially correct the very poor binding of RE-1 and RF-2 to L7/L12-depleted ribosomes (15). To investigate the biological significance and function of RF-3 in stop codon recognition, polypeptide release, and cell g...

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl-tRNA hydrolysis activity and enhanced codon-ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where -1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol-1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.

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