Sally A. Mannering

Three, four or more: the translational stop signal at length

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: codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, approximately 40,000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor 3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.

HIDDEN INFIDELITIES OF THE TRANSLATIONAL STOP SIGNAL

Publisher Summary This chapter provides evidence that there is a subtle layer of cellular regulation in which translational stop signals play a part and for which the relative strength of the signals is critical. Specific signals for the termination of protein synthesis were predicted from the studies of nonsense mutations. Such mutations were associated with the appearance of amino-terminal fragments of the affected gene products, indicating that protein synthesis had been prematurely terminated. A simultaneous investigation with the bacteriophage T4rII gene also identified UAA and UAG as translational stop codons. The historical development of ideas and discoveries related to the functioning of the genetic code has molded the perception about the translational stop signal as a triplet. However, experiments investigating the effects of the surrounding nucleotide sequence and the suppression of stop signals by suppressor or noncognate tRNAs suggested hidden infidelities in the signal. There is much evidence suggesting that the efficiency of translational stop signals can be influenced in cis by the surrounding sequence of mRNA. There are some examples of suppression of translational stop signals by naturally occurring tRNAs that may be physiologically significant. Special protein factors are involved in the recognition of translational stop signals as part of the termination machinery. Their existence was initially suggested by in vitro studies indicating a factor in cell-free extracts apart from tRNA, ribosomes, or elongation factors that influenced peptide release.

Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex.

There have been contrasting reports of whether the positioning of a translational stop signal immediately after a start codon in a single oligonucleotide can act as a model template to support efficient in vitro termination. This paradox stimulated this study of what determines the constraints on the positioning of the components in the termination complex. The mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, release factor 2, was stimulated with ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes which have a higher affinity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA. An additional triplet stop codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already within it. This suggests that in an initiation complex an adjoining start/stop signal is not positioned to support efficient decoding by release factor unless it is separated from the start codon. Site-directed crosslinking from mRNAs to components of the termination complex has shown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release factor, and presumably the orientation of the signal to the factor.

THE STOP SIGNAL CONTROLS THE EFFICIENCY OF RELEASE FACTOR­ MEDIATED TRANSLATIONAL TERMINATION

There are three important steps in protein synthesis where signals in the mRNA are critical for a successful outcome, namely the production of a functional protein. First the information in the nucleic acid which is to be translated into an amino acid sequence is signalled by successive triplet sense codons, second the frame is set by one sense codon, the initiation codon, which acts as the start of translation of the encoded information, and third the end of the information frame also has to be marked by a specific signal. The use of a range of different signals to mark each of these steps allows for differences in the efficiency with which different proteins are produced. In this review the focus is on the signal that marks the end of the frame, the translational termination signal. For a long time it was thought that termination would be the least interesting phase of protein synthesis but it has subsequently been found to have unexpected dimensions, providing a substratum of cellular regulation. The translational stop signal should now be thought of as a full stop in the large majority of cases, but as a pause in a fundamentally important minority of cases where alternative genetic events can occur.