Kim K. McCaughan

Molecular mimicry in the decoding of translational stop signals

Molecular mimicry was a concept that was revived as we understood more about the ligands that bound to the active center of the ribosome, and the characteristics of the active center itself. It has been particularly useful for the termination phase of protein synthesis, because for many years this major process seemed not only to be out of step) with the initiation and elongation phases but also there were no common features of the process between eubacteria and eukaryotes. As the facts that supported molecular mimicry emerged, it was seen that the protein factors that facilitated polypeptide chain release when the decoding of an mRNA was complete had common features with the ligands involved in the other phases. Moreover, now common features and mechanisms began to emerge between the eubacterial and eukaryotic RFs and suddenly there seemed to be remarkable synergy between the external ligands and commonality in at least some features of the mechanistic prnciples. Almost 10 years after molecular mimicry took hold as a framework concept, we can now see that this idea is probably too simple. For example, structural mimicry can be apparent if there are extensive conformational changes either in the ribosome active center or in the ligand itself or, most likely, both. Early indications are that the bacterial RF may indeed undergo extensive conformational changes from its solution structure to achieve this accommodation. Thus, as important if not more important than structural and functional mimicry among the ligands, might be their accomodation of a common single active center made up of at least three parts to carry out a complex series of reactions. One part of the ribosomal active center is committed to decoding, a second is committed to the chemistry of putting the protein together and releasing it, and a third part, perhaps residing in the subdomains, is committed to binding ligands so that they can perform their respective single or multiple functions. It might be more accurate to regard the decoding RF as the cuckoo taking over the nest that was crafted and honed through evolution by another, the tRNA. A somewhat ungainly RF, perhaps bigger in dimensions than the tRNA, is able, nevertheless, like the cuckoo, to maneuvre into the nest. Perhaps it pushes the nest a little out of shape, but is still able to use the site for its own functions of stop signal decoding and for facilitating the release of the polypeptide. The term molecular mimicry has been dominant in the literature for a period of important advances in the understanding of protein synthesis. When the first structures of the ribosome appeared, the concept survived and was seen to be valid still. Now, we are at the stage of understanding the more detailed molecular interactions between ligands and the rRNA in particular, and how subtle changes in localized spatial orientations of atoms occur within these interactions. The simplicity of the original concept of mimicry will inevitably be blurred by this more detailed analysis. Nevertheless, it has provided a significant set of principles that allowed development of experimental programs to enhance our understanding of the dynamic events at this remarkable active site at the interface between the two subunits of this fascinating cell organelle, the ribosome.

Functional characterization of yeast mitochondrial release factor 1.

The yeast Saccharomyces cerevisiaemitochondrial release factor was expressed from the clonedMRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.

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.

Translational Stop Signals: Evolution, Decoding for Protein Synthesis and Recoding for Alternative Events

Termination or translational stopping involves a close relationship between the ribosome, the mRNA, and the polypeptide chain release factors. The discovery of an array of alternative events occurring at stop codons in the mRNA has focussed attention on how the decoding mechanism discriminates between simple ‘stop’ signals and alternative events outside the normal constraints of the genetic code (Tate and Brown, 1992). This latter phenomenon has been recently called ‘recoding’ and the signals ‘recoding signals’ (Gesteland et al., 1992). Is the normal role of the stop codon merely overriden by the recoding signals or does the stop component of such a signal contribute to a finely tuned regulation at sites where the alternative events occur? These questions demand a re-examination of how a stop signal recognition mechanism might have evolved, first for its function in translational stopping, and second for a possible role in gene regulation.

The ribosomal binding domain for the bacterial release factors RF-1, RF-2 and RF-3

The Escherichia coli ribosomal proteins, L7/L12, are dominant over L11 in modulating the binding of RF‐1 and RF‐2 to ribosomes. The elevated activity of RF‐2 on L11‐lacking ribosomes over those containing L11 is abolished by IgG against L7/L12 or by removing the L7/L12 proteins. Adding back L7/L12 restores the original phenotype. The stimulatory factor, RF‐3, is active on ribosomes depleted of L7/L12 but on those which lack L11 the stimulatory effects are less pronounced or often not seen. RF‐3 cannot restore activity with RF‐1 or RF‐2 to ribosomes lacking both these sets of proteins. The stimulatory effects of an absence of either L11 or RF‐3 on the activity of RF‐2 are not additive or synergistic.