What is the mechanism by which mammalian ribosomes are released from the mRNA following termination of translation?

The strong physical, behavioural, and mental similarity of identical twins shows that it is our genetic material that largely specifies what we are. This genetic material is our DNA, which can be regarded as an exceedingly long 'tape' (e.g. an enormous video tape) with some 3 billion bits of information, coding for the numerous different types of protein which carry out most of our bodily functions. The DNA can be thought of as the 'master tape', an archive of thousands of normal length videos joined together. The process of decoding this information in the DNA involves first making a working copy of one of these videotapes in the archive. This is then decoded by a small particle known as a ribosome, which essentially carries out an analogous function to that of a video recorder (VCR) as it decodes the information as pictures. These 'biological videos' are similar to real videos in so far as the part of the tape with the pictorial information is preceded by a short leader length that has no information, and ends with a trailer that is likewise meaningless. We know that our biological VCR (the ribosome) finds the point where the true information starts by searching or scanning in fast-forward mode through the leader. However, when the ribosome-like VCR has reached the end of the pictorial information, it does not scan or try to read the meaningless trailer. Instead, as soon as the pictorial information has come to an end, the biological video is essentially ejected from the biological VCR. While we understand how this ejection mechanism works in the case of the bacterial 'VCR', we know absolutely nothing about how the mammalian eject mechanism works, except that it seems to be completely different from the bacterial mechanism. The aim of this project is to find out how the mammalian 'biological VCR' ejects the biological video cassette.

It is universally agreed that when mammalian polyribosomes are incubated in the presence of an inhibitor of initiation, the ribosomes run off the mRNA (i.e. they are released from the mRNA following termination) and accumulate as 80S monomeric ribosomes. However, we know absolutely nothing about the mechanism of this release, or what protein factors are required. This represents the most significant complete gap in our understanding of the mechanism of mRNA translation in eukaryotes. It is known that in eubacteria the ribosome release is catalysed by RRF (ribosome release factor) together with elongation factor eEF2 and GTP. However, no RRF orthologue has been found in eukaryotic cytoplasm. One of the reasons why we are so ignorant of the mechanism of ribosome release from the mRNA following termination at a stop codon is that the assays for the release of the peptide by the termination factors use short oligonucleotides as mRNA-mimics, which readily dissociate spontaneously from the ribosome. One of the 3 sub-projects in this proposal will use longer RNAs (>35 nt.) with a zero-length open reading frame (e.g. ..AUGUAA..), which should remain associated with the ribosome following hydrolysis of the P-site (formyl)Met-tRNAi. Another sub-project will follow the approach that was successfully used to identify and purify eubacterial ribosome release factor (RRF). This examines the release of ribosomes from the mRNA following premature pseudo-termination by puromycin, which leaves the ribosome in precisely the same state as bona fide termination at a stop codon: an empty A-site and a deacylated tRNA in the P-site (or the P/E site in the hybrid states model). The project will examine whether ribosome release from the mRNA in either of these situations is promoted by any combination of the termination factors eRF1 and eRF3 plus eEF2 (and GTP). In the event that no combination of these factors effects ribosome release, we will examine whether post-ribosomal supernatant contains an additional protein capable of promoting such release, and, if so, we will attempt to purify any such activity and characterise it. There are indications in the literature that after puromycin-mediated pseudo-termination of translation, ribosomes may reinitiate translation close to the position on the mRNA where the puromycin intervention occurred. The implication is that either there is no ribosome release factor that can function in this situation, or that any such factor is inefficient. As a corollary of our search for a mammalian RRF, we will examine whether post-puromycin reinitiation really does occur, and, if so, we will attempt to measure the efficiency of any such in-frame reinitiation, and address the question of whether reinitiation also occurs in the other two reading frames.