Natural examples of non-triplet decoding during protein synthesis have been discovered in a wide variety of genes. A recurring theme in these cases is the use of flanking sequence elements to perturb tRNA:mRNA interaction resulting in high-level alternate decoding events such as frameshifting, read-through, and the newly defined event termed """"""""hopping"""""""". The goal of this project is to use these sequence elements as tools for identifying the ribosomal and cellular factors that interact with them. An exemplary case of hopping occurs in bacteriophage T4 gene 60 where ribosomes bypass a 50 nt. internal gap within the coding region. Mutational analysis of gene 60/lacZ fusions has shown that bypass occurs with nearly 100% efficiency in Escherichia coli, and inactivation of the bypass mechanism by alteration of the gene has revealed a complex group of sequence elements required for bypass. In the first part of the project, we will make gene 60 containing constructs and perform protein sequencing studies to further define the mRNA components involved concentrating on the reasons for apparent optimal distance for by-pass. In the second part, we will use a variety of host mutations that reduce bypass in a reversion analysis that may identify the components that normally mediate functions that these elements disturb. Similar, though subtly different, pseudoknots stimulate retroviral stop codon read-through and at least certain cases of retroviral frameshifting. In a third part of the project, the differences between the pseudoknots, and the mechanism(s) involved will be investigated. The pol gene of the Foamy virus sub-family of Retroviruses is in the +1 frame with respect to its gag gene, unlike the other Retrovirus sub-families. In a fourth part, in collaboration with others, we propose to investigate the mode of ribosome entry to the Simian Foamy virus pol gene. The frameshifting utilized by several bacterial insertion sequences has retroviral-like frameshift site sequences, but with interesting differences. In a fifth part, in collaboration with others, we propose to investigate the frameshifting utilized by IS911.
Showing the most recent 10 out of 85 publications
