Department of Pathology

Dr Ian Brierley

Research description

Cryo-EM image of ribosomal-stalling at a frameshift-promoting RNA pseudoknot (purple). Shown are 40S subunit (yellow), bent tRNA (green) and associated eEF2 (red; Namy 2006 Nature 441 p244ff).

We study the regulation of virus gene expression at the level of protein synthesis, especially the phenomenon of ribosomal frameshifting. During translation, ribosomes generally stay in register and decode each codon triplet by triplet. However, some viral mRNAs have embedded signals that instruct ribosomes to change register, ie: to frameshift, at a defined position and to continue translation in an overlapping reading frame. Frameshift signals are exploited by many viruses to co-ordinate gene expression from overlapping coding sequences. The best studied examples come from the retroviruses. Here, the coding region for the enzymatic functions of the virus (pol) often overlaps the upstream structural protein coding sequences (gag) and is in the -1 reading frame. Expression of the pol gene requires a -1 frameshift at the end of gag and the Pol protein is produced as a fusion with the Gag protein. Our aim is to understand the frameshift process with a view to inhibiting it and thus to prevent virus replication. Many other RNA viruses have been shown to utilise a frameshift strategy in their replication cycle and there are emerging examples in cellular genes. A closely related translational phenomenon, termination codon suppression (readthrough), has also been described. Here, viral mRNA signals cause the misreading of stop codons, and an amino acid is inserted instead, at a certain frequency, resulting in an elongated polypeptide.

We are studying frameshifting and readthrough using coronaviruses and retroviruses as model systems. A frameshift signal has two elements, a "slippery sequence" where the ribosome enters the new reading frame, followed by an mRNA structure, often an RNA pseudoknot. A bipartite signal is also employed in readthrough but here, the pseudoknot induces suppression of an upstream stop codon. Our aim is to determine the mechanism by which these signals induce the ribosome to change frame/misread the stop codon. Further, we are interested in gaining an understanding of RNA pseudoknot structure and function. The pseudoknot motif has been described in all cellular RNAs described to date and pseudoknots are clearly key regulatory RNA elements. Projects are underway to study:

(1) Transacting factors, both protein and RNA, which influence frameshifting/readthrough (2)the structure of frameshift and readthrough stimulatory RNAs (3) ribosome conformation during frameshifting/readthrough (4) the effect on virus replication of altering frameshift/readthrough efficiencies and (5) ribosomal pausing at RNA pseudoknots and its relevance to frameshifting/readthrough, including ribosomal profiling of virus-infected cells to identify sites of ribosomal pausing in vivo.