Translational stop signals are defined in the genetic code as UAA, UAG and UGA, although the mechanism of their decoding via protein factors is clearly different from that of the other codons. There are strong biases in the upstream and downstream nucleotides surrounding stop codons. Experimental tests have shown that termination-signal strength is strongly influenced by the identity of the nucleotide immediately downstream of the codon (+4), with a correlation between the strength of this four-base signal and its occurrence at termination sites. The +4 nucleotide and other biases downstream of the stop codon may reflect sites of contact between the release factor and the mRNA, whereas upstream biases may be due to coding restrictions, with the release factor perhaps recognizing the final tRNA and the last two amino acids of the polypeptide undergoing synthesis. This means that the translational stop signal is probably larger than the triplet codon, but its exact length will be clearer when it is known which nucleotides are in direct contact with the release factor. Ultimately it will be defined exactly when a crystal structure of the release factor with its recognition substrate becomes available.

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.

The observations that the Escherichia coli release factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop signals, stimulated us to determine whether there was a more extended termination signal for RF2 recognition. Analysis of the 3' contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element. Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10). Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2. A strong selection bias for particular bases in the +4 to +6 positions of certain E. coli UGANNN termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo termination signal strength. These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence termination signal decoding efficiency.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.

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