The role of mRNA secondary structures in programmed termination codon readthrough

Whilst a cell's proteins are encoded in DNA (the genetic material), they are synthesised by an organelle called the ribosome through an intermediate molecule, messenger RNA (mRNA), that is transcribed from DNA. The appropriate end of the mRNA is fed into the ribosome which moves along until a triplet start signal in the mRNA is recognised. At this point, amino acid biosynthesis starts and as each subsequent triplet nucleotide 'code' is decoded, one amino acid is added to a growing amino acid chain. The ribosome continues along the mRNA until it recognises a triplet stop signal, at which point translation normally terminates and the completed amino acid polymer, the protein, is released. However in some instances, the stop signal is not recognised correctly (it is said to be 'leaky'), and instead, an amino acid is added and protein synthesis continues. This is called stop codon readthrough and is the subject of our investigation. Stop codon readthrough is used by cells and viruses to make, at a certain frequency, an elongated version of a protein, which has altered properties. We are interested in determining how the structure of the mRNA in the vicinity of the 'leaky' stop codon can cause the ribosome to fail to recognise the stop efficiently. At present, we know little about the RNA structures that promote readthrough. In this project, we will examine the structures from three different readthrough signals and look for common features to give us clues as to how the ribosome is being mislead. A deeper knowledge of this process will provide insights into the biology of gene expression and our knowledge of ribosome function.

This project will examine the role of RNA secondary structures in programmed termination codon readthrough. The aim is to determine how the stimulatory RNAs of murine leukemia virus (MuLV), Colorado tick fever virus (CTFV) segment 9 and Drosophila headcase act to promote readthrough. We will begin by determining the secondary structure of the CTFV and headcase signals using RNA structure probing techniques and site-directed mutagenesis coupled with functional assays. Subsequently, we will investigate the interaction of the stimulatory RNAs with the ribosome and with other cellular proteins. Ribosome-RNA interactions will be probed by UV crosslinking and in ribosomal pausing assays. We will test specifically the interaction between ribosomal protein S15a and the stimulatory RNAs. S15a is close to the ribosomal helicase and could interact with the stimulatory RNA as the termination codon enters the decoding site. Cellular proteins that can interact with readthrough-promoting RNA structures will be identified by RNA affinity column chromatography and mass spectroscopy. Involvement in readthrough will be tested using siRNAs.