Dr. Joseph Wade
Dr Joseph T. Wade is an Assistant Professor at the School of Public Health in the New York State Department of Health. His research interests include microbial gene expression in eukaryotes and prokaryotes, specifically transcription. He has successfully established an independent laboratory that applies microarray technology to bacterial gene expression.
He provided insight into the role of 5'UTRs in gene expression and suggested changes to the protocol of our experimentation process. In his opinion, the end goal of this research must be to predict the effect on gene expression based on the primary structure alone. However, optimistically we were given to understand that we were far from achieving that. He suggested that in creating a library for 5'UTRs, we should have specific sequences based on or closely derived from the naturally occurring 5’ UTRs in Escherichia coli, instead of preparing a randomized series of sequences. This gave us a great starting point for our experiments and changed our thoughts on the experimental design.
This approach is based on the fact that trans-acting molecular interactions would be exceedingly rare in the case of a randomized library approach. In contrast, cis-acting intramolecular interactions would be far more common. Both types of factors play an essential role in translation and transcription. Upon being asked whether the results of a particular 5’ UTR with GFP as a marker would be translatable to other systems as well, he responded that there needs to be a standard set of variables that would not be changed to interpret the data. Although, this depends on whether the goal is to identify the role of the base pairs close to the Shine-Dalgarno sequence or to identify the effects of 5’ UTRs in general, which have a much greater degree of variation. If 5’ UTRs are characterized using more than one gene, it is quite possible that some trans-acting factors could selectively regulate the expression of one and not the other. If a difference in the expression levels is discovered, further investigation would be required to identify the cause of the difference in the expression levels. This may be achieved by mutating RNA sequences and testing the expression levels for mutated sequences. Making chimeric fusions by cloning the sequence of a 5’ UTR to the SD of another would also be insightful, given that mutations are tested too.
The secondary structure of the mRNA is a function of the sequence and temperature, and one may be able to manipulate temperature conditions to reduce or enhance the secondary structure. Osmolarity, he mentioned, affects base pairing, but not much research has been done on it. There are genes characterized by their change in expression due to osmolarity. This may be correlated with the secondary structure of 5’ UTR. Pathogenic organisms use osmolarity and temperature to detect whether they are in a host, as mammalian hosts have a higher body temperature. The RNA thermometers of these pathogens then help conditionally express genes that initiate infection.
